U.S. patent application number 11/640800 was filed with the patent office on 2009-03-19 for bioreactive agents.
This patent application is currently assigned to The Trustees of Boston University. Invention is credited to Jerzy Olejnik, Kenneth J. Rothschild, Sanjay M. Sonar.
Application Number | 20090075253 11/640800 |
Document ID | / |
Family ID | 26994551 |
Filed Date | 2009-03-19 |
United States Patent
Application |
20090075253 |
Kind Code |
A1 |
Rothschild; Kenneth J. ; et
al. |
March 19, 2009 |
BIOREACTIVE AGENTS
Abstract
This invention relates to agents and conjugates that can be used
to detect and isolate target components from complex mixtures such
as nucleic acids from biological samples, cells from bodily fluids,
and nascent proteins from translation reactions. Agents comprise a
detectable moiety bound to a photoreactive moiety. Conjugates
comprise agents coupled to substrates by covalent bounds which can
be selectively cleaved with the administration of electromagnetic
radiation. Targets substances labeled with detectable molecules can
be easily identified and separated from a heterologous mixture of
substances. Exposure of the conjugate to radiation releases the
target in a functional form and completely unaltered. Using
photocleavable molecular precursors as the conjugates, label can be
incorporated into macromolecules, the nascent macromolecules
isolated and the label completely removed. The invention also
relates to targets isolated with these conjugates which may be
useful as pharmaceutical agents or compositions that can be
administered to humans and other mammals. Useful compositions
include biological agents such as nucleic acids, proteins, lipids
and cytokines. Conjugates can also be used to monitor the pathway
and half-life of pharmaceutical composition in vivo and for
diagnostic, therapeutic and prophylactic purposes. The invention
also relates to kits comprised of agents and conjugates that can be
used for the detection of diseases, disorders and nearly any
individual substance in a complex background of substances.
Inventors: |
Rothschild; Kenneth J.;
(Newton, MA) ; Sonar; Sanjay M.; (Thane, IN)
; Olejnik; Jerzy; (Brookline, MA) |
Correspondence
Address: |
MEDLEN & CARROLL, LLP
Suite 350, 101 Howard Street
San Francisco
CA
94105
US
|
Assignee: |
The Trustees of Boston
University
|
Family ID: |
26994551 |
Appl. No.: |
11/640800 |
Filed: |
December 18, 2006 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10396095 |
Sep 8, 2003 |
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11640800 |
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09504001 |
Feb 14, 2000 |
6589736 |
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10396095 |
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08479389 |
Jun 7, 1995 |
6057096 |
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09504001 |
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08345807 |
Nov 22, 1994 |
5986076 |
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08479389 |
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08240511 |
May 11, 1994 |
5643722 |
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08345807 |
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Current U.S.
Class: |
435/6.13 |
Current CPC
Class: |
C07D 405/12 20130101;
C12Q 1/6823 20130101; C07D 311/16 20130101; C07D 495/04 20130101;
G01N 33/5306 20130101; A61K 38/00 20130101; C07H 19/04 20130101;
C12Q 1/6876 20130101; C12P 21/00 20130101; C07H 21/00 20130101;
C07C 205/45 20130101; C07C 205/57 20130101; A61K 41/0042 20130101;
C12Q 2523/319 20130101; G01N 33/58 20130101; C07D 207/404 20130101;
C12Q 1/6823 20130101; C07K 1/1077 20130101; C07K 1/13 20130101;
G01N 33/532 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Goverment Interests
RIGHTS IN THE INVENTION
[0002] This invention was made with United States Government
support under grant number EM4727-03, awarded by the National
Institutes of Health, and grant number DAAL03-92-G-0172, awarded by
the Army Research Office, and the United States Government has
certain rights in the invention
Claims
1. (canceled)
2. (canceled)
3. (canceled)
4. (canceled)
5. (canceled)
6. A method, comprising: a) providing i) nucleic acid, ii) a
conjugate, comprising a substrate covalently coupled to a
detectable moiety through a cleavable linker, said substrate
selected from the group consisting of nucleosides and nucleotides,
said detectable moiety having the property of fluorescence, and
iii) a polymerase; and b) mixing said nucleic acid and said
polymerase in the presence of said conjugate under conditions such
that said conjugate is incorporated into said nucleic acid to
produce labeled nucleic acid.
7. The method of claim 6, wherein said nucleic acid is RNA.
8. The method of claim 6, wherein said nucleic acid is DNA.
9. The method of claim 6, wherein said detectable moiety is
selected from the group consisting of rhodamines and
fluoresceins.
10. The method of claim 6, wherein said substrate is a nucleotide
selected from the group consisting of NTP, dNTP and ddNTP
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a continuation-in-part of U.S.
patent application Ser. No. 08/240,511, filed May 11, 1994.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] This invention relates to agents and conjugates used in the
detection and isolation of targets from heterologous mixtures.
Agents comprise a detectable moiety bound to a photoreactive
moiety. Conjugates comprise agents which are coupled to substrates
by one or more covalent bonds. These bonds can be easily and
selectively cleaved or photocleaved with the application of
electromagnetic radiation. Substrates which may be coupled to
agents include amino acids, peptides, proteins, nucleotides,
nucleic acid primers for PCR reactions and lipids. The invention
also relates to rapid and efficient methods for the detection and
isolation of targets, such as cells, nucleic acids and proteins,
and to kits which contain these components.
[0005] 2. Description of the Background
[0006] Basic scientific techniques including some of the major
breakthroughs in molecular biology, chemistry and medicine have
certain features in common. Two of these features are the specific
detection and isolation of individual components from complex
mixtures. For example, electrophoresis and chromatography are each
widely utilized procedures to detect or isolate macromolecules from
biological samples. These procedures take advantage of unique or
identifiable molecular properties of the components to be isolated
such as charge, hydrophobicity and molecular weight, to
characterize and identify macromolecules. Depending on their method
of isolation, macromolecules isolated can often be utilized as
products in downstream processes.
[0007] Some of the more useful detection and isolation procedures
take advantage of physical properties of the element of interest,
the substrate, or of molecules which can be easily attached to
substrates. One of the most widely used of these properties is
radioactivity and radioactive labeling with radionuclides. For the
most part, substances are not naturally radioactive and can be
labeled with radioactive atoms, referred to as radionuclides, and
detected using standard and well-known radiographic procedures.
Radioactive elements are detectable because they emit large amounts
of energy in the form of alpha, beta or gamma rays as they decay.
Radioactivity is generally useful for labeling because the label is
not affected by the physical state or chemical combination of the
substance to be labeled. In addition, the specific radiation
emitted can be identified by the nature of the radiation (e.g.
.alpha., .beta. or .gamma.), its energy and the half-life of the
process. Targets can be identified in complex mixtures from the
radiation profile emitted. Further, radioactively labeled
substances can be followed radiographically in chemical pathways
and biological systems.
[0008] Unfortunately, radioactivity is a hazard to both human
health and the environment. The protection which must be afforded
each worker is substantial. Special laboratory procedures,
dedicated facilities and equipment, detailed record keeping and
special training of laboratory personnel are all required for the
safe use of radionuclides. Production of radioactive reagents is
also very expensive as is safe disposal which drives up the cost of
all experiments involving radioactive agents. Further, under
present guidelines, all users of radioactivity require specialized
supervision and federal regulations must be strictly and carefully
adhered to requiring an enormous amount of record keeping.
[0009] Radioactive labeling methods also do not always provide a
means of isolating products in a form which can be further
utilized. The presence of radioactivity compromises utility for
further biochemical or biophysical procedures in the laboratory and
in animals. This is clear in the case of in vitro or in vivo
expression of proteins biosynthetically labeled with radioactive
amino acids or tagged with other radioactive markers. The harm or
at least potential harm of the radioactivity outweighs the benefits
which might be produced by the protein composition.
[0010] Disposal of radioactive waste is also of increasing concern
both because of the potential risk to the public and the lack of
radioactive waste disposal sites. In addition, the use of
radioactive labeling is time consuming, in some cases requiring as
much as several days for detection of the radioactive label. The
long time needed for such experiments is a key consideration and
can seriously impede research productivity. While faster methods of
radioactive detection are available, they are expensive and often
require complex image enhancement devices.
[0011] There are many other detectable physical properties which
can exist in chemicals and chemical moieties that can be used to
detect and isolate substances. One of these physical properties is
the property of luminescence which includes the phenomena of
fluorescence and phosphorescence. Fluorescent chemicals emit
radiation due to the decay of the molecule which has been excited
to a higher electronic state due to the absorption of radiation.
Phosphorescent molecules can emit radiation for a much longer time
intervals. Detection of the specific wavelength of radiant energy
emitted allows for the detection of targets which may be associated
with the luminescent chemicals.
[0012] Bioluminescence is rapidly becoming a widely used method for
labeling many different types of compounds. Basically, a reduced
substrate is reacted with oxygen and converted into an oxidized
product with an elevated or excited electronic state. The excited
molecule decays to the ground state and in the process, emits
photons of light. This process has been found to occur in several
strains of bacteria and fungi, in marine invertebrates such as
sponges, and in shrimp and jellyfish. Bacteria which emit light are
often found living symbiotically with fish in special luminescent
organs. A wide variety of terrestrial organisms such as earthworms,
centipedes and insects also possess bioluminescent properties.
[0013] One group of compounds which undergo oxidation with the
emission of light are referred to as luciferins although their
individual structure may vary. The oxidized products are termed
oxy-luciferins and the enzymes which catalyze the process
luciferases. The overall process is endothermic requiring chemical
energy stored in one to two molecules of adenosine triphosphate
(ATP) per photon of light produced. Two types of luciferase systems
that have been widely used in molecular biology are the bacterial
system (Vibrio harveyi or V. fischeri) and the firefly system
(Photinus pyralis).
[0014] Other labels which impart detectable properties to a
substrate include chemicals with a unique absorption spectrum,
electron spin resonance spectrum, optical activity, Raman spectrum
or resonance Raman spectrum. Such labels are widely used in many
fields including medicine, molecular biology and chemistry. For
example, the visible or infrared absorption spectrum of a molecule
often constitutes a unique fingerprint which allows the molecule to
be identified even in the presence of a complex mixture. In the
case of visible absorption, the molecule absorbs radiant energy
over a specific wavelength range because of the presence of an
excited electronic state of the molecule whose energy of transition
from the ground state falls in the range 1.5-3 eV. In the case of
infrared absorption, bands are detected due to the excitation of
vibrational modes of the molecule. The frequency of these bands
provides information about the presence or absence of
characteristic molecular groups such as disulfides, carbonyls and
aromatic groups.
[0015] In another application of the spectroscopic properties of
molecules, nuclear magnetic resonance (NMR) spectroscopy has been
extensively used to identify specific molecules in a mixture. The
nuclei of atoms, such as protons in hydrogen atoms, which possess a
net magnetic moment, will align when placed into a magnetic field
with that field and will precess about that field with a frequency
(the Lamar frequency) dependant on the individual properties of the
particle. To determine the NMR spectrum, a sample of protons is
placed within a strong magnetic field and irradiated with a range
of radio frequencies at a 90.degree. angle with respect to the main
field. This treatment causes all the protons in the sample to
absorb energy at their characteristic frequency, flipping their
magnetic orientations 90.degree. with respect to their original
state. After the applied field is switched off, the molecules
gradually relax to precess about the main field. Receiver coils
which surround the sample detect the frequencies of precessing
spins as a set of oscillating electric currents which constitute
the NMR signal.
[0016] All of these methods suffer from similar disadvantages. For
the most part, targets do not have unique detectable properties
such as inherent radioactivity or fluorescence. Labels must be
attached which are themselves detectable and therefore make the
target detectable. However, the labeling process can result in a
labeled product that is in some way permanently damaged. For
example, fluorescent chemicals can be extremely toxic to cells.
Long term exposure can result in a high degree of cell death.
Often, the labeling compound may have detrimental effects on a
target's structure or activity. Protein structure is often
adversely affected by the attachment of a detectable chemical
moiety. Labeling of nucleic acids can interfere with their ability
to be translated, transcribed by polymerases or interact with DNA
binding proteins. In most cases, the chemical moiety must be
removed. Further, the methods for removal of these chemical
moieties which have detectable physical properties often result
directly in alteration of the molecule or cell death.
[0017] There are a number of procedures, both complex and simple,
which have been used to selectively detect and isolate target
substrates. One procedure which has revolutionized and greatly
accelerated the detection and identification of nucleic acids is
polymerase chain reaction (PCR) technology. The principle concept
of PCR is the rapid, large-scale amplification of unique or even
non-unique nucleic acid sequences in biological samples. Using
labeled primers with specific or random sequences, the genetic code
of very small quantities of nucleic acids can be detected,
amplified in number and subsequently characterized through
repetitive polymerization events. Although the nucleic acids formed
are new, the sequence of the original sample is maintained and can
be easily determined. As a nucleic acid sequence is the biological
code for the construction of virtually all proteins, the origin,
evolutionary age, structure and composition of nearly any
biological organism or sample can be determined from knowledge of
the sequence. The procedure has been proved useful in molecular and
evolutionary biology, and has demonstrated applications in the
detection, treatment and prevention of diseases and disorders in
humans.
[0018] PCR technology, although revolutionary, carries with it the
same limitations as many conventional detection and isolation
procedures. Label which has been incorporated into primers and
ultimately newly formed nucleic acids must be removed. This
process, when possible, is fairly time consuming and often results
is modification or destruction of the nucleic acid.
[0019] Another example of a process to render a substance
specifically detectable is to use binding molecules which have a
particular affinity for selected other molecules as occurs between
binding of an antigen to an antigen-specific antibody. These
chemical pairs, sometimes referred to as coupling agents, have been
used extensively in detection and isolation procedures. Normally
one of the molecules in this pair is immobilized on an affinity
medium such as used in chromatographic packing material or a
magnetic bead and used in the isolation of the target molecule.
Some of the more useful coupling agents are biotin and avidin or
the related protein, streptavidin. These agents have been used in
many separation techniques to facilitate isolation of one component
or another from complex mixtures
[0020] Biotin, a water-soluble vitamin, is used extensively in
biochemistry and molecular biology for a variety of purposes
including macromolecular detection, purification and isolation, and
in cytochemical staining. Biotin also has important applications in
medicine in the areas of clinical diagnostic assays, tumor imaging
and drug delivery, and is used extensively in the field of affinity
cytochemistry for the selective labeling of cells, subcellular
structures and proteins.
[0021] Biotin's utility stems from its ability to bind strongly to
the tetrameric protein avidin, found in egg white and the tissues
of birds, reptiles and amphibians, or to its chemical cousin,
streptavidin, isolated from the bacterium Streptomyces. Typically,
biotin or a derivative of biotin is first bound directly to a
target molecule, such as a protein or oligonucleotide, or to a
probe using specific chemical linkage. The interaction of the
linked biotin with either streptavidin or avidin conjugated to an
affinity medium such as magnetic or sepharose beads is then used in
the isolation of the target molecule. Alternatively, the
interaction of the covalently linked biotin with avidin or
streptavidin conjugated to an enzyme such as horseradish peroxidase
(HRP) which catalyzes a chromogenic reaction is used for detection
of the target molecule. Macromolecules that have been isolated
using biotin-avidin technology are shown in Tables 1 and 2.
TABLE-US-00001 TABLE 1 Macromolecules Isolated by Direct
Biotinylation Biotinylated Targets Elution Conditions References
Membrane proteins acetate, pH 4 1, 2 and glycoproteins Antibodies
low pH 3 Enzymes non-physiological 4 t-RNA 6M guanidine-HCl, 5 pH
2.5 rRNA 70% formic acid 6 nucleosomes SS-reduction of 7, 8
cleavable biotin DNA non-physiological 9 1. G. A. Orr, J. Biol.
Chem. 256: 761, 1981. 2. C. A. Beard et al., Mol. Biochem.
Parasitol. 16: 199, 1985. 3. E. A. Bayer et al., FEBS Lett. 68:
240, 1976. 4. C. S. Chandler et al., Biochem. J. 237: 123, 1986. 5.
T. R. Broker et al., Nucl. Acids Res. 5: 363, 1978. 6. D. J.
Eckermann et al., Eur. J. Biochem. 82: 225, 1978. 7. M. L. Shimkus
et al., Proc. Natl. Acad. Sci. USA 82: 2593, 1986. 8. M. L. Shimkus
et al., DNA 5: 247, 1986. 9. P. L. Langer et al., Proc. Natl. Acad.
Sci. USA 78: 6633, 1981.
TABLE-US-00002 TABLE 2 Biological Materials Isolated Using
Biotinylated Binding Molecules Binding Target Molecules Molecules
Elution Conditions Refs. Glycoproteins conconavalin A 2% SDS 10
Membrane Antigens antibody SDS (boiling) 11 Estrogen Receptor
estradiacetate, estradiol 12 Insulin Receptor insulin acetate, pH
5.0 13, 14 biotin Opoid Receptor enkephalin enkephalin 15 Human B
antigen selection by FACS 16 lymphocytes Lymphocyte monoclonal
Mechanical 17, 18, 19 subpopulations antibody agitation,
erythrocyte lysis Plasmid DNA DNA 0.1 M NaOH 20 Spliceosomes RNA
90.degree. C. in SDS 21 Recombinant DNA Cleavable biotin 22
Plasmids Heat, low ionic strength and phenol 10. J. W. Buckie et
al., Anal. Biochem. 156: 463, 1986. 11. T. V. Updyke et al., J.
Immunol. Methods 73: 83, 1984. 12. G. Redeulih et al., J. Biol.
Chem. 260: 3996, 1985. 13. F. M. Finn et al., Proc. Natl. Acad.
Sci. USA 81: 7328, 1984. 14. R. A. Kohanski et al., J. Biol. Chem.
260: 5014, 1985. 15. H. Nakayama et al., FEBS Lett. 208: 278, 1986.
16. P. Casali et al., Sci. 234: 476, 1986. 17. J. Wormmeester et
al., J. Immunol. Methods 67: 389, 1984. 18. P. J. Lucas et al., J.
Immunol. Methods 99: 123, 1987. 19. R. J. Berenson et al., J.
Immunol. Methods 91: 11, 1986. 20. H. Delius et al., Nucl. Acids
Res. 13: 5457, 1985. 21. P. J. Grabowski et al., Sci. 233: 1294,
1986. 22. B. Riggs et al., Proc. Natl. Acad. Sci. USA 83: 9591,
1986.
[0022] While the utility of biotin continues to grow, there still
exists major drawbacks in the use of biotin-streptavidin technology
for many applications. This problem stems from the high affinity
between biotin and streptavidin, precisely the molecular
characteristic which makes it most useful. Once a target molecule
or cell is isolated through the streptavidin-biotin interaction,
release of the target molecule requires disruption of this
interaction. Dissociation of biotin from streptavidin requires very
harsh conditions such as 6-8 molar (M) guanidinium-HCl, pH 1.5.
Such conditions also denature, and thereby inactivate, most
proteins and destroy most cells.
[0023] For example, a biotin derivative containing a
N-hydroxysuccinimide ester group is commonly used to link biotin
through an amide bond to proteins and nucleic acids. Selective
cleavage of this linkage disrupts similar native chemical bonds in
associated molecules. Biotin is also often used in the isolation of
specific cells from a heterogeneous mixture of cells by binding a
biotinylated antibody directed against a characteristic cell
surface antigen. The interaction of the biotinylated antibody with
streptavidin-coated magnetic beads or sepharose particles can then
be used effectively to isolate target cells. Disruption of the
antibody-antigen interaction normally requires exposure of cells to
conditions such as low pH or mechanical agitation which are adverse
to the cell's survival. In general, recovery of the target in a
completely unmodified form is not possible.
[0024] Once the biotinylated DNA is bound to streptavidin it can
only be released with extreme difficulty. Many diverse methods to
remove the streptavidin molecule have been suggested including
digestion by proteinase K (M. Wilchek and E. A Bayer, Anal.
Biochem. 171:1, 1988). Proteinase K also digests nearby proteins
and does a fairly poor job of completely digesting the
streptavidin. Significant amounts of the streptavidin molecules
remain attached, and further, removal of streptavidin does not
release the biotin. Further, biotinylated DNA interferes with
subsequent use in a variety of methods including transformation of
cells and hybridization based assays used for detection of genetic
diseases.
[0025] The essentially irreversible binding of biotin and
streptavidin is also a serious limitation for the performance of
multiple or sequential assays to detect a specific type of
biomolecule, macromolecular complex, virus or cell present in a
single sample. Normally, only a single assay can be performed
because the enzyme detection system is streptavidin-based and
streptavidin remains firmly bound to the biotinylated target or
target probe. While different chromogenic systems for detection are
available, they are only of limited applicability in situations
where large numbers of probes are needed.
[0026] An additional problem in the use of biotin-avidin technology
is the presence of endogenous biotin, either free or complexed to
other molecules, inside the sample to be purified or assayed. In
this case, the endogenous biotin can result in the isolation or
detection of non-target molecules. This can be a particularly
severe problem in cases where a high signal-to-noise ratio is
needed for accurate and sensitive detection.
[0027] To remove biotin from an attached molecule, several
chemically cleavable biotin derivatives have been produced.
Immunopure NHS--SS-biotin (Pierce Chemical; Rockford, Ill.)
consists of a biotin molecule linked through a disulfide bond and
an N-hydroxysuccinimide ester group that reacts selectively with
primary amines. Using this group, NHS--SS-biotin can be linked to a
protein and then the biotin portion removed by cleaving the
disulfide bond with thiols. This approach is of limited use since
thiols normally disrupt native disulfide bonds in proteins.
Furthermore, the cleavage still leaves the target cell or molecule
modified since the spacer arm portion of the complex is not removed
and the cleaving buffer must be eliminated from the sample.
[0028] One method for removal of biotin is the use of
disulfide-based cleavable biotins. However, the cleaved molecules
possess a reactive sulfhydryl group which has a strong tendency to
form disulfide bonds with other components of the mixture.
Functional activity of these substances containing sulfhydryl
groups is severely compromised. Typically, activity of such protein
is decreased or eliminated and such nucleic acids will no longer
hybridize rendering them useless for cloning. This method is also
slow and requires the preparation of complex solutions.
[0029] An additional limitation of biotin-avidin technology is the
difficulty of developing automated systems for the isolation and/or
detection of targets due to the problems of releasing the target
from the biotin-avidin binding complex. This requires addition of
specific chemical reagents and careful monitoring of the
reactions.
[0030] Biotin-avidin technology has been combined with PCR
techniques for the detection and isolation of nucleic acids and
specific sequences. However, there still remains a fundamental
problem which relates to the difficulty of removing the
incorporated biotin. This is normally not possible using
conventional biotins without irreversibly altering the structure of
the DNA. As discussed, biotinylation can interfere with subsequent
application of biotinylated probes as well as alter the properties
of the PCR product.
[0031] PCR products that contain biotinylated nucleotides or
primers which are required for isolation cannot be used in
conjunction with biotinylated hybridization probes. The presence of
biotin on the PCR product cause false signals from the avidin based
enzyme-linked detection system. Biotin incorporation into DNA
interferes with strand hybridization possibly due to the spacer
arms linking the nucleotides to the biotin molecules. Further, PCR
products that are biotinylated are not suitable material for
cloning. PCR products which contain biotinylated nucleotides are
difficult to analyze. Incorporation of biotinylated nucleotides
into DNA causes a retardation of mobility during gel
electrophoresis in agarose. This mobility shift renders
characterization of PCR products difficult. As proper DNA-DNA
hybridization is the basis for sensitive and accurate
characterization and sensitive assays, biotin-avidin binding
systems are seriously disadvantaged.
[0032] Other coupling partners which can be used to detect and
isolate target substances are cell adhesion molecules (CAMs). One
of the well characterized types is the endothelial cell adhesion
molecule, LEC-CAM (leukocyte endothelial cell-cell adhesion
molecule), now called selectin. This molecule selectively binds to
leukocytes. Its natural function is to facilitate the transport of
leukocytes through an endothelial layer of cells such as
postcapillary venules to sites of inflammation or tissue damage.
There are many of these adhesion molecules which have been
identified in humans and other mammals that range in binding
specificity from the very general to the highly specific. These
include the endothelial cell adhesion ligands ICAM-1, VCAM-1 and
ELAM-1, the .beta.-integrins which consists of a family of three
proteins LFA-1, Mac-1, VLA4, MO-1 and p150/95, carbohydrate binding
CAMs that appear on endothelial cells, platelets, and leukocytes,
and the cadherins, calcium dependent CAMs present on most cells.
Attachment of these molecules or the creation of fused proteins
containing adhesion domains can be used to facilitate isolation and
detection of binding partners. However, once binding has occurred,
complex, expensive and time consuming biochemical manipulations and
sometimes fairly harsh chemical treatments are necessary to
dissociate the molecules. Further, application of these molecules
for general use is limited as binding partners must be located for
each target of interest.
[0033] Other coupling partners include nucleic acids and nucleic
acid binding proteins, lipids and lipid binding proteins, and
proteins or specific domains which have a particular affinity for
each other. These coupling partners suffer from similar drawbacks
as the biotin-avidin system and the adhesion molecules.
[0034] Another fairly ubiquitous method of detection and isolation
is gel electrophoresis. In this process, a uniform matrix or gel is
formed of, for example, polyacrylamide, to which is applied an
electric field. Mixtures applied to one end of the gel will migrate
through the gel according to their size and interaction with the
electric field. Mobility is dependent upon the unique
characteristics of the substance such as conformation, size and
charge. Mobilities can be influenced by altering pore sizes of the
gel, such as by formation of a concentration or pH gradient, or by
altering the composition of the buffer (pH, SDS, DOC, glycine,
salt). One- and two-dimensional gel electrophoresis are fairly
routine procedures in most research laboratories. Target substances
can be purified by passage through and/or physical extraction from
the gel.
[0035] Methods for the detection and isolation of targets
substances also include centrifugation techniques such as
equilibrium-density-gradient centrifugation. This process is based
on the principal that under high centrifugal forces, stable
gradients will be established in salt solutions. Mixtures subjected
to high speed centrifugation will segregate individual components
according to their specific densities. Although useful, all of
these procedures are more quantitative than qualitative.
[0036] A major advance in detection and isolation methodology was
the advent of liquid chromatography. Chromatography, and in
particular column chromatography, comprises some of the most
effective and flexible purification methods available. Common to
most procedures is the use of open cylinders containing a hydrated
matrix material. Some of the typical matrix materials which are
presently used, for example, in gel filtration, affinity
chromatography and ion exchange chromatography, include sepharose
(bead formed gel prepared from agarose: Pharmacia Biotech;
Piscataway, N.J.), sephadex (a bead-formed gel prepared by
cross-linking dextran with epichlorohydrin: Pharmacia Biotech;
Piscataway, N.J.) and sephacryl (covalently cross-linked allyl
dextran with N,N'-methylene bisacrylamide: Pharmacia Biotech;
Piscataway, N.J.). Basically, a heterogenous sample or mixture is
applied to the top of the column followed with a suitable buffer.
Substances within the mixture display differential migration
through the column in relation to other materials within the sample
and is collected in fractions at the other end of the column.
Fractions are individually analyzed for the presence of target and
positive fractions pooled.
[0037] Alternatively, target in the sample may selectively bind to
the column material in the presence of buffer a process known as
affinity chromatography. After binding, unbound material is removed
by continuously washing the column with buffer. Target molecules
are subsequently released from the column by application of an
elution buffer which causes dissociation. Fractions are collected
as they elute off of the column and collected. In gel-exclusion
chromatography, a cross-linked dextran is utilized as column matrix
material. Cross-linking can be varied to alter the effective pore
size of the column material and the dextran can be coupled to a
wide variety of chemical moieties to selectively capture target.
Ion-exchange chromatography takes advantage of the fact that
targets, for example proteins, can differ enormously in their
affinity for positive or negative charges on column materials. The
affinity of a material for a target is proportional to the salt
concentration of the buffer. By raising or lowering the salt
concentration, it is possible to change affinity of target to
column material.
[0038] Affinity column chromatography makes use of chemical groups
that have special attraction to the targets of interest. For
example, enzymes preferentially bind to certain naturally
associated cofactors. Column materials with attached cofactors will
selectively bind to such target enzymes. Enzyme purification
becomes a relatively simple and straightforward matter. In a
similar fashion, enzyme-specific antibodies can be coupled either
covalently or non-covalently to a column matrix. The unique
affinity of an antibody for its target antigen allows for the
selective removal of target from a heterologous mixture of
substances. Detection and isolation is again a fairly simple
matter.
[0039] Two relatively well-established procedures, high-performance
liquid chromatography (HPLC) including reverse-phase HPLC and
size-exclusion HPLC, and the more recent technique fast-performance
liquid chromatography (FPLC) which can handle larger sample volumes
than HPLC, is based on standard chromatographic techniques, but
using extremely high pressures (5,000 to 10,000 psi and more). Due
to the higher pressures, finer column materials can be utilized and
separations can be performed faster and with better resolution.
[0040] Although chromatography is an invaluable tool it too has its
limits. Materials to be separated must be solubilized into a
suitable buffer which will not adversely affect the column.
Further, substrate mixtures and targets must be capable of passing
through a column matrix in a reasonable period of time. Although
HPLC can sometimes shorten this time period, only small quantities
can be detected and the high pressures can damage isolated column
material.
SUMMARY OF THE INVENTION
[0041] The present invention overcomes the problems and
disadvantages associated with current strategies and designs and
provides new methods for the detection and isolation of molecules
from complex mixtures.
[0042] One embodiment of the invention is directed to bioreactive
agents comprising a detectable moiety bonded to a photoreactive
moiety wherein the photoreactive moiety contains at least one group
capable of covalently bonding to a substrate to form a conjugate
that can be selectively photocleaved to release said substrate.
Detectable moieties should have a selectively detectable physical
property such as fluorescence, absorption or an ability to
specifically bind to a coupling agent such as avidin or
streptavidin, antibodies, antigens or binding proteins. The
photoreactive moiety should be capable of forming one or more
covalent bonds with a chemical group of a substrate. Those covalent
bonds may be cleaved or photocleaved with the electromagnetic
radiation releasing the substrate.
[0043] The bioreactive agent may have a chemical structure selected
from the group consisting of:
##STR00001##
wherein X is selected from the group consisting of a halogen,
N.sub.2, CH.sub.2-halogen, --N.dbd.C.dbd.O, --N.dbd.C.dbd.S,
--S--S--R, NC.sub.2H.sub.4, --NC.sub.4H.sub.2O.sub.2, --OH,
--NHNH.sub.2, --OP(OR.sub.3)N(R.sub.4)R.sub.5 and --OCO-G, wherein
G is selected from the group consisting of a halogen, N.sub.3,
O-esters and N-amides; R.sub.1, R.sub.2, R.sub.3, R.sub.4 and
R.sub.5, are selected from the group consisting of hydrogen,
alkyls, substituted alkyls, aryls and substituted aryls,
--CF.sub.3, --NO.sub.2, --COOH and --COOR, and may be the same or
different; A is a divalent functional group selected from the group
consisting of --O--, --S-- and --NR.sub.1; Y comprises one or more
polyatomic groups which may be the same or different; V comprises
one or more optional monoatomic groups which may be the same or
different; Q comprises an optional spacer moiety; m1 and m2 are
integers from 0-5 and may be the same or different; and D comprises
a detectable moiety which is distinct from R.sub.1-R.sub.5.
[0044] Another embodiment of the invention is directed to
conjugates comprising a bioreactive agent photocleavably coupled to
a substrate wherein said agent comprises a detectable moiety bonded
to a photoreactive moiety, wherein said conjugate can be
selectively cleaved with electromagnetic radiation to release said
substrate. Suitable substrates which can be coupled to the
bioreactive agent include proteins, peptides, amino acids, amino
acid analogs, nucleic acids, nucleosides, nucleotides, lipids,
vesicles, detergent micells, cells, virus particles, fatty acids,
saccharides, polysaccharides, inorganic molecules, metals, and
derivatives and combinations thereof. Substrates may be
pharmaceutical agents such as cytokines, immune system modulators,
agents of the hematopoietic system, chemotherapeutic agents,
radio-isotopes, antigens, anti-neoplastic agents, recombinant
proteins, enzymes, PCR products, receptors, hormones, vaccines,
haptens, toxins, antibiotics, nascent proteins, cells, synthetic
pharmaceuticals and derivatives and combinations thereof.
[0045] Conjugates may have a chemical structure selected from the
group consisting of:
##STR00002##
[0046] wherein SUB comprises a substrate; R.sub.1, and R.sub.2 are
selected from the group consisting of hydrogen, alkyls, substituted
alkyls, aryls, substituted aryls, --CF.sub.3, NO.sub.2, --COOH and
--COOR, and may be the same or different; A is a divalent
functional group selected from the group consisting of --O--, --S--
and --NR.sub.1; Y comprises one or more polyatomic groups which may
be the same or different; V comprises one or more optional
monoatomic groups which may be the same or different; Q comprises
an optional spacer moiety; m1 and m2 are integers between 1-5 which
can be the same or different; and D comprises a detectable moiety
which is distinct from R.sub.1 and R.sub.2.
[0047] Another embodiment of the invention is directed to
pharmaceutical compositions comprising the conjugate plus a
pharmaceutically acceptable carrier such as water, an oil, a lipid,
a saccharide, a polysaccharide, glycerol a collagen or a
combination thereof. Pharmaceuticals may be used in the prophylaxis
and treatments of diseases and disorders in humans and other
mammals.
[0048] Another embodiment of the invention is directed to methods
for isolating targets from a heterologous mixture. Briefly, a
conjugate is created by coupling a bioreactive agent to a substrate
by a covalent bond which is selectively cleavable with
electromagnetic radiation wherein the bioreactive agent is
comprised of a photoreactive moiety bonded to a detectable moiety.
The conjugate is contacted to the heterologous mixture to couple
substrate to one or more targets. The coupled conjugate is
separated from the mixture and treated with electromagnetic
radiation to release the substrate, and the targets isolated. This
method can be used to isolate targets such as immune system
modulators, cytokines, agents of the hematopoietic system,
proteins, hormones, gene products, antigens, cells, toxins,
bacteria, membrane vesicles, virus particles, and combinations
thereof from heterologous mixtures such as biological samples,
proteinaceous compositions, nucleic acids, biomass, immortalized
cell cultures, primary cell cultures, vesicles, animal models,
mammals, cellular and cell membrane extracts, cells in vivo and
combinations thereof.
[0049] Another embodiment of the invention is directed to target
molecules isolated by the above methods which may be used in
pharmaceutical compositions or other compositions and mixtures for
industrial applications.
[0050] Another embodiment of the invention is directed to methods
for isolating targets from a heterologous mixture. A conjugate is
created comprising a bioreactive agent coupled to a substrate by a
covalent bond which is selectively cleavable with electromagnetic
radiation, wherein said bioreactive agent is comprised of a
photoreactive moiety bonded to a detectable moiety and the
substrate is a precursor of the target. The conjugate is contacted
with the heterologous mixture to incorporate substrate into
targets. The incorporated conjugate is separated from the mixture,
treated with electromagnetic radiation to release the substrate,
and the targets isolated. This method is useful for the detection
and isolation of nascent proteins, nucleic acids and other
biological substances.
[0051] Another embodiment of the invention is directed to methods
for isolating targets from a heterologous mixture. A conjugate is
created which is comprised of a bioreactive agent coupled to a
receptor by a covalent bond which is selectively cleavable with
electromagnetic radiation, wherein said bioreactive agent is
comprised of a photoreactive moiety bonded to a detectable moiety.
The conjugate is contacted with the heterologous mixture to couple
receptor to targets and the coupled receptor-targets separated from
the mixture. The separated conjugate is treated with
electromagnetic radiation to release the receptor and the targets
isolated.
[0052] Another embodiment of the invention is directed to methods
for isolating target cells from a heterologous mixture. A conjugate
is created comprising a bioreactive agent coupled to a cell
receptor by a covalent bond which is selectively cleavable with
electromagnetic radiation, wherein the bioreactive agent is
comprised of a photoreactive moiety bonded to a detectable moiety.
The conjugate is contacted with the heterologous mixture to couple
receptor to target cells. The coupled conjugate is separated from
the mixture and treated with electromagnetic radiation to release
the substrate. Target cells are then easily isolated such as by
automation.
[0053] Another embodiment of the invention is directed to methods
for creating a photocleavable oligonucleotide. A conjugate is
created comprising a bioreactive agent coupled to a phosphoramidite
which may be a purine-phosphoramidite or a
pyrimidine-phosphoramidite. The oligonucleotide is synthesized
using photocleavable phosphoramidites. The process can be performed
manually or automated to be carried out by an oligonucleotide
synthesizer.
[0054] Another embodiment of the invention is directed to methods
for determining an in vivo half-life of a pharmaceutical in a
patient. A conjugate is formed by coupling the pharmaceutical to a
bioreactive agent with a covalent bond that can be selectively
cleaved with electromagnetic radiation, wherein said bioreactive
agent comprises a photoreactive moiety bonded to a detectable
moiety. The conjugate is administered to the patient and at least
two or more biological samples are removed from the patient at
various times after administration of the conjugate. The samples
are treated with electromagnetic radiation to release the
pharmaceutical from the bioreactive agent and the amount of the
bioreactive agent in the biological samples determined. The in vivo
half-life of the pharmaceutical can be determined.
[0055] Another embodiment of the invention is directed to methods
for the controlled release of a substrate into a medium. A
conjugate comprised of a bioreactive agent coupled to the substrate
by a covalent bond which can be selectively cleaved with
electromagnetic radiation is created wherein the bioreactive agent
is comprised of a detectable moiety bonded to a photoreactive
moiety. The conjugate is bound to a surface of an article which is
placed into the medium. The surface of the article is exposed to a
measured amount of electromagnetic radiation for the controlled
release of the substrate into the medium.
[0056] Another embodiment of the invention is directed to methods
for detecting a target molecule in a heterologous mixture. A
conjugate is formed by coupling a substrate to a bioreactive agent
with a covalent bond that is selectively cleavable with
electromagnetic radiation, wherein the bioreactive agent is
comprised of a detectable moiety bonded to a photoreactive moiety.
The conjugate is contacted with the heterologous mixture to couple
substrate to one or more target molecules. Uncoupled conjugates are
removed and the coupled conjugates are treated with electromagnetic
radiation to release the detectable moiety. The released detectable
moiety can now be easily detected.
[0057] Another embodiment of the invention is directed to methods
for detecting a target molecule in a heterologous mixture. A
conjugate, comprising a substrate coupled to a bioreactive agent,
is formed and contacted with a heterologous mixture to couple a
conjugate to one or more target molecules. Uncoupled conjugates are
removed and the coupled conjugates are treated with electromagnetic
radiation to release substrate. Released substrate is detected and
can be further isolated.
[0058] Another embodiment of the invention is directed to methods
for the isolation of a PCR product. A bioreactive agent is
conjugated to one or more oligonucleotide primers with a covalent
bond that is selectively cleavable with electromagnetic radiation,
wherein the bioreactive agent is comprised of a detectable moiety
bonded to a photoreactive moiety. A nucleic acid sequence is PCR
amplified with the conjugated primers. The amplified sequences are
isolated and subsequently treated with electromagnetic radiation to
release the bioreactive agent.
[0059] Another embodiment of the invention is directed to methods
for treating a disorder by the controlled release of a therapeutic
agent at a selected site. A conjugate is formed by bonding a
bioreactive agent to the therapeutic agent with a bond that is
selectively cleavable with electromagnetic radiation, wherein the
bioreactive agent is comprised of a directable moiety bonded to a
photoreactive moiety wherein the directable moiety has an affinity
for the selected site. The conjugate is administered to a patient
having the disorder. The selected site is subjected to a measured
amount of electromagnetic radiation for the controlled release of
the therapeutic agent to treat the disorder.
[0060] Another embodiment of the invention is directed to kits for
detecting a disorder in biological samples containing conjugates
comprised of a bioreactive agent covalently bonded to a diagnostic
agent having an affinity for an indicator of the disorder in the
biological sample, wherein the covalent bond is selectively
cleavable with electromagnetic radiation.
[0061] Another embodiment of the invention is directed to kits
comprising a bioreactive agent covalently bonded to an
oligonucleotide. The photocleavable oligonucleotide may be
double-stranded or single-stranded and may possess restriction
enzyme recognition sites useful in cloning and other procedures in
molecular biology.
[0062] Other embodiments and advantages of the invention are set
forth, in part, in the description which follows and, in part, will
be obvious from this description or may be learned from the
practice of the invention.
DESCRIPTION OF THE FIGURES
[0063] FIG. 1 Examples of photocleavable agents.
[0064] FIG. 2 Photocleavable biotins with various photoreactive
moieties.
[0065] FIG. 3 Schematic representation of photocleavable biotin
[0066] FIG. 4 Mechanism of photocleavage in 2-nitrobenzyl-based
systems.
[0067] FIG. 5 Synthesis of photocleavable biotin
[0068] FIG. 6 Chemical variations of photocleavable agents.
[0069] FIG. 7 Photolysis of PCBs.
[0070] FIG. 8 PCB conjugates.
[0071] FIG. 9 Possible amino acid linkages of PCB.
[0072] FIG. 10 (A) Aminoacylation of tRNA, and (B) a comparison
between enzymatic and chemical aminoacylation.
[0073] FIG. 11 The four basic steps in the isolation of pure
substrate using PCB.
[0074] FIG. 12 Method for the detection of target protein and its
antibody using PCB.
[0075] FIG. 13 PCB-phosphoramidites and PCB-nucleotides.
[0076] FIG. 14 Two methods (A and B) for the isolation of
PCR-product using PCB.
[0077] FIG. 15 Comparison of methods for the construction of a cDNA
library (A) with PCB and (B) without PCB.
[0078] FIG. 16 PCB lipids.
[0079] FIG. 17 Immunoselective cell separation using PCB.
[0080] FIG. 18 Synthesis of photocleavable biotin-NHS ester,
compound 18.
[0081] FIG. 19 Synthesis of photocleavable biotin-NHS ester,
compound 25.
[0082] FIG. 20 Method for sequential ELISA using PCB.
[0083] FIG. 21 Synthesis of photocleavable coumarin.
DESCRIPTION OF THE INVENTION
[0084] As embodied and broadly described herein, the present
invention is directed to agents and conjugates used in the
detection and isolation of targets such as chemicals,
macromolecules, cells and any identifiable substance from a
mixture. Agents comprise a detectable moiety bound to a
photoreactive moiety. Conjugates comprise agents which are coupled
to substrates by one or more covalent bonds which, by the presence
of the photoreactive moiety, are selectively cleavable with
electromagnetic radiation The invention is also directed to methods
for the isolation and detection of targets using these agents and
conjugates, to kits which utilize these methods for the detection
of diseases and disorders in patients, and to methods for the
detection and isolation of nearly any substance from a heterologous
mixture.
[0085] There are many methods currently available for the detection
and isolation of a desired substance or target from a complex
mixture. Most of these methods require the specific labeling of the
substance or target to be detected, detection of that label and
subsequent removal of label from target. Although straightforward,
current detection and isolation methodologies possess a number of
problems. For example, it is often difficult to specifically attach
target with label. Affinity of label for target may be low,
suitable points of attachment may not be available on specific
substances and the label and the target may simply be chemically or
physically incompatible. In addition, label may hinder or
completely destroy the functional activity of the target
frustrating the purpose of isolation. Isolated targets are
unavoidably contaminated or inactivated due to the presence of a
toxic or damaging label. A typical example of this sort of problem
is the isolation of cells bound with biotin after selection by
coupling to streptavidin. The powerful affinity of biotin for
streptavidin makes the isolation procedure relatively
straightforward and specific, however, the isolated cells are often
dead and dying due to the toxic effects of the coupling agents or
the harsh isolation procedures. This is also true when attempting
to isolate active proteins from biological samples and other
complex mixtures for later use. The presence of label may denature
or render the protein product inactive or simply unacceptable for
in vivo use under current FDA standards and guidelines. Removal of
the agent sometimes overcomes these problems, however, methods to
separate and remove label from target are generally rather harsh,
take a significant amount of time, effort and expense, and, for the
most part, result in fairly low yields of the final product.
Viability and functional activity of the target is often severely
impaired and is often destroyed.
[0086] The invention overcomes these problems by providing
detectable, bioreactive agents which can detect and isolate
targets. Agents of the invention comprise a detectable moiety and a
photoreactive moiety, and can be covalently coupled to a variety of
target substrates. A covalent bond between agent and substrate can
be created from a wide variety of chemical moieties including
amines, hydroxyls, imidazoles, aldehydes, carboxylic acids, esters
and thiols. Agent-substrate combinations are referred to herein as
conjugates. Through the presence of the detectable moiety,
conjugates can be quickly and accurately detected and target
isolated. Further, these conjugates are selectively cleavable which
provides unique advantages in isolation procedures. Substrate can
be separated from agent quickly and efficiently. Complex technical
procedures and highly trained experts are not required. New
attachment and separation procedures do not need to be developed
for every new target to be isolated. Following isolation, it is a
relatively simple matter to treat the conjugate with
electromagnetic radiation and release the substrate. Released
substrate is preferably functionally active and structurally
unaltered. Nevertheless, minor chemical alterations in the
structure may occur depending on the point of attachment. It is
generally preferred that such alterations not effect functional
activity. However, when functional activity does not need to be
preserved, such changes are of no considerations and may even be
useful to identify and distinguish targets isolated by methods of
the invention.
[0087] Targets, as referred to herein, are those substances being
identified, characterized or isolated using the agents, conjugates
and methods of the invention. Substrates, as referred to herein,
are those substances which are covalently attached to the
bioreactive agent. Substrates may also be referred to as targets
when the target being identified specifically binds to the
bioreactive agent.
[0088] One embodiment of the invention is directed to a bioreactive
agent comprising a detectable moiety bonded to a photoreactive
moiety wherein the photoreactive moiety contains at least one group
capable of covalently bonding to a substrate to form a conjugate.
The resulting conjugate can be selectively cleaved to release said
substrate or, alternatively, to release any chemical group or agent
of the conjugate. Cleavage, as referred to herein, is by
photocleavage or a cleavage event triggered by the application of
radiation to the conjugate. The radiation applied may comprise one
or more wavelengths from the electromagnetic spectrum including
x-rays (about 0.1 nm to about 10.0 mm; or about 10.sup.18 to about
10.sup.16 Hz), ultraviolet (UV) rays (about 10.0 nm to about 380
nm; or about 8.times.10.sup.16 to about 10.sup.15 Hz), visible
light (about 380 nm to about 750 nm; or about 8.times.10.sup.14 to
about 4.times.10.sup.14 Hz), infrared light (about 750 nm to about
0.1 nm; or about 4.times.10.sup.14 to about 5.times.10.sup.11 Hz),
microwaves (about 0.1 cm to about 100 cm; or about 10.sup.8 to
about 5.times.10.sup.11 Hz), and radio waves (about 100 cm to about
10.sup.4 m; or about 10.sup.4 to about 10.sup.8 Hz). Multiple forms
of radiation may also be applied simultaneously, in combination or
coordinated in a step-wise fashion. Radiation exposure may be
constant over a period of seconds, minutes or hours, or varied with
pulses at predetermined intervals.
[0089] Typically, the radiation source is placed at a specified
distance from the conjugate to be irradiated. That distance may be
empirically determined or calculated from the energy loss produced
between the source and the target and the amount of energy emitted
by the source. Conjugate may be in solution or attached to a solid
support which may be a type of glass, ceramic, polymer or
semiconductor surfaces. Typical solid supports are nitrocellulose
membranes, agarose beads, magnetic beads coated with streptavidin,
semiconductor surfaces and resins. Preferably, the radiation
applied is UV, visible or IR radiation of the wavelength between
about 200 nm to about 1,000 nm, more preferably between about 260
nm to about 600 nm, and more preferably between about 300 nm to
about 500 nm. Radiation is administered continuously or as pulses
for hours, minutes or seconds, and preferably for the shortest
amount of time possible to minimize any risk of damage to the
substrate and for convenience. Radiation may be administered for
less than about one hour, preferably less for than about 30
minutes, more preferably for less than about ten minutes, and still
more preferably for less than about one minute. Visible, UV and IR
radiation are also preferred as all three of these forms of
radiation can be conveniently and inexpensively generated from
commercially available sources.
[0090] The power density or intensity of light per area necessary
to selectively cleave the covalent bond is very small which makes
the photocleavable process practical. Maximization of efficiency
also minimizes exposure time necessary to achieve selective
cleavage and provide a minimum of undesirable background
effects.
[0091] One part of the bioreactive agent is the detectable moiety.
The detectable moiety is a chemical group, structure or compound
that possesses a specifically identifiable physical property which
can be distinguished from the physical properties of other
chemicals present in the heterologous mixture. Fluorescence,
phosphorescence and luminescence including electroluminescence,
chemiluminescence and bioluminescence are all detectable physical
properties not found in most substances, but known to occur or to
be inducible in others. For example, reactive derivatives of
dansyl, coumarins, rhodamine and fluorescein are all inherently
fluorescent when excited with light of a specific wavelength and
can be specifically bound or attached to other substances. Coumarin
has a high fluorescent quantum yield, higher than even a dansyl
moiety, and facilitates detection where very low levels of target
that are being sought. Coumarin is structurally similar to
tryptophan, which can be useful in for example in the translation
of nascent proteins with non-native amino acids. It may also be
useful to combine certain detectable moieties to facilitate
detection or isolation. Preferably the detectable moiety is a
fluorescent compound and the preferred fluorescent compounds are
listed in Table 3, all of which are commercially available (Sigma
Chemical; St. Louis, Mo.).
TABLE-US-00003 TABLE 3 Fluorescent Labeling Compounds
4-acetamido-4'-isothiocyanatostilbene-2-2'-disulfonic acid
7-amino-4-methylcoumarin (AMC) 7-amino-4-trifluoromethylcoumarin
N-(4-anilino-1-naphthyl) maleimide 4',6-diamidino-2-phenylindole
(DAPI) 5-(4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF)
4,4'-dilsothiocyanatostilbene-2,2'-disulfonic acid
tetramethylrhodamine isothiocyanate (TRITC) quinolizino fluorescein
isothiocyanate (QFITC) dansyl chloride eosin isothiocyanate
erythrosin B fluorescamine fluorescene fluorescein derivatives
4-methylumbelliferone o-phthaldialdehyde rhodamine B rhodamine B
derivatives rhodamine 6G rhodamine 123 sulforhodamine B
sulforhodamine 101 sulforhodamine 101 acid chloride
[0092] Luminescence can also be induced in certain chemicals
referred to as luciferins. Energetic molecules such as ATP supply
chemical energy for catalytic activities of luciferase enzymes
causing the luciferins to emit light. Reagents for both the
bacterial luciferase system (Vibrio harveyi or V. fischeri) and the
firefly luciferase system (Photinus pyralis) are available from a
variety of commercial sources (e.g. Sigma Chem. Co; St. Louis,
Mo.).
[0093] Preferably, the luminescent agent has a high quantum yield
of fluorescence at a wavelength of excitation different from that
used to perform the photocleavage. Upon excitation at such
wavelengths, the agent is detectable at low concentrations either
visually or using conventional luminescence detectors and
fluorescence spectrometers. Electroluminescence, produced by agents
such as ruthenium chelates and their derivatives, or agents that
possess nitroxide moieties and similar derivatives are preferred
when extreme sensitivity is desired (J. DiCesare et al.,
BioTechniques 15:152-59, 1993). These agents are detectable at the
femtomolar ranges and below.
[0094] Application of an electric field will also induce a
detectable response in certain chemicals due to a net electric
charge which induces the substance to migrate in an electric field.
Magnetism may also be a detectable property if a magnetized
substance such as iron or another magnetized metal is or is
associated with the detectable moiety.
[0095] Other forms of detectable physical properties include an
identifiable electrical polarizability, electron spin resonance and
Raman scattering. Agents may also undergo a chemical, biochemical,
enzymatic, electrochemical or photochemical reaction such as a
color change in response to external electromagnetic fields or the
introduction of other substances. Such electromagnetic fields and
substances may be a catalyst or another reactant molecule that
allows for detection of the bioreactive agent or transforms the
agent into a detectable moiety.
[0096] All of these labeling agents can be specifically detected
using the appropriate detector or detection system such as a
spectrometer or electrophoretic or chromatographic systems. At
times, it may be preferable to have a visually discernable
detection system such as one that will trigger a photoelectric cell
or one that can be detected and recorded manually. Spectrometers
including absorption and fluorescence spectrometers are very
sensitive detectors of specific energy of absorptions or emissions
from many detectable moieties. Detection and sorting of target may
be automated as in the case of fluorescence activated cell sorters
(FACS) which detect and isolate cells that possess a fluorescent
label. Targets may be detected and sorted manually as can be done
quite simply with magnetized conjugates using a magnet.
[0097] Additional physical properties which can be easily and
accurately detected include chromaticism (e.g. violet=about 400-430
nm, blue=about 450-500 nm, green=about 550 nm, yellow=about 600 nm,
orange=about 650 nm, red=about 700-750 nm), electromagnetic
absorbance, enzyme activity or the ability to specifically bind
with a coupling agent. Useful coupling agents include biotin,
avidin, streptavidin, nucleic acids, nucleic acid and lipid binding
proteins, haptens, antibodies, receptors, carbohydrates,
immunogenic molecules, and derivatives and combinations thereof.
The detectable moiety may have a combination of these properties
allowing its selection from a wide variety of background materials.
Some examples of the chemical structures of photocleavable agents
of the invention are depicted in FIG. 1.
[0098] Another preferred detectable moiety is a coupling agent and
the preferred coupling agent is biotin or a biotin derivative.
Biotin-containing bioreactive agents are referred to herein as
photocleavable biotins or PCBs. The binding between the egg-white
protein avidin, a tetrameric protein found in avian eggs with the
water soluble vitamin, biotin, is one of the strongest interactions
known in biology having an association constant (K.sub.a) of about
10.sup.15M.sup.-1, exceeding that of antibody-antigen interactions
(M. Wilchek and E. A. Bayer, Methods Enzymol. 184, 1990). The
bacterial counterpart to avidin is streptavidin, found in
Streptomyces avidinii, which is slightly more specific for biotin
than avidin. This strong interaction, along with the ability to
covalently link biotin to a variety of substrates including
proteins, nucleic acids, lipids, and receptor ligands such as
neuropeptides and hormones, has resulted in a vast array of uses
for these coupling agents all of which can be improved or enhanced
with the use of PCB.
[0099] A wide variety of biotinyl moieties can be used to form a
PCB molecule. Biotin (C.sub.10H.sub.16N.sub.2O.sub.3S) has a
molecular weight of 244.31 daltons and is comprised of a ring
linked to an alkyl chain terminated by a carboxyl group. Numerous
modifications can be made to the biotin moiety which involve
changes in the ring, spacer arm and terminating group, all of which
still exhibit a high affinity for streptavidin, avidin and their
derivatives. Examples of photocleavable biotins that can be
designed based on various photoreactive moieties are depicted in
FIG. 2.
[0100] The detection and isolation of chemical, biochemical and
biological materials using the interaction between biotin and
streptavidin is normally based on the immobilization of avidin or
streptavidin to a surface (e.g. membranes, gels, filters,
microtiter wells, magnetic beads). To that surface is applied a
solution containing biotin coupled to targets which then bind to
the streptavidin-coated surface. Biotin-containing target molecules
can be isolated and non-biotinylated components washed away.
Alternatively, biotinylated target molecules can be separated from
a heterogeneous mixture using streptavidin-containing affinity
columns. Biotinylated macromolecules including nucleic acids (DNA
or RNA), proteins and protein-containing complexes, and even cells
whose surface has been biotinylated or bound to a biotinylated
antibody can be detected and isolated with these techniques.
[0101] As stated, biotin can be coupled to a wide variety of
molecules including proteins, carbohydrates and nucleic acids. The
availability of biotin derivatives has expanded this range even
further. For example, biotin derivatives have been prepared with
functionalities which are reactive towards amines, phenols,
imidazoles, aldehydes, carboxylic acids and thiols. Biotin can also
be incorporated into proteins, DNA and RNA by first attaching the
biotin to building blocks of macromolecules such as amino acid or
nucleotides which can be directly attached to these molecules or
incorporated during their synthesis by chemical or enzymatic
means.
[0102] Unlike conventional biotins, photocleavable biotins enable
one to release or elute the bound substrate from the immobilized
avidin, streptavidin or their derivatives in a completely
unmodified form. This is extremely useful and an important
improvement over existing biotins for a number of reasons.
Biotinylation of the target material can impede its subsequent use
or characterization. Biotinylation of a protein can alter its
activity, electrophoretic mobility, ability to bind a substrate,
antigenicity, ability to reconstitute into a native form and
ability to form multisubunit complexes. In contrast, using
photocleavable biotin, once the biotin is photocleaved from the
protein or protein/binding complex, all the native properties and
function will be restored to its native form for further use and
characterization. Listed in Table 4 are some of the substrates to
which a photocleavable agent such as PCB can be linked.
TABLE-US-00004 TABLE 4 Chemical linkage of Photocleavable Biotins
with different molecules Resulting linkage Molecule or Functional
Group Reactive moiety and reaction Assemblage on the Molecule on
the PCB conditions Amino acids, Amino group or NHS-ester Amide
linkage proteins, enzymes R--NH.sub.2 or antibodies Amino acids,
R--OH chloroformate Ester linkage proteins, enzymes carboxylic acid
or antibodies Amino acids, R--COOH Reaction with Ester linkage
proteins, enzymes parent alcohol or antibodies (DCC coupling)
Nucleotides, Aromatic amines chloroformate Amides RNA or DNA
molecules Carbohydrates Sugar hydroxy chloroformate Ester linkage
RNA for R--OH ribonucleotides Nucleotide Phosphate groups
diazoethane Phosphate ester phosphoramidites Lipids/ R--NH.sub.2
Chloroformate amide Phosphatidyl NHS-ester serine Carbohydrates
Sugar hydroxyl Chloroformate Ester linkage
[0103] There are a number of chemical moieties available in
bioreactive agents and conjugates of the invention. For example, an
NHS-ester functionality introduced in PCB is highly reactive and
can selectively react with aliphatic amino groups that are present
in proteins. Another example is a phosphoramidite moiety which is
highly reactive and can selectively react with hydroxyl groups of
nucleic acids. In cases where chemical moieties like carboxyl
(--COOH) or phosphate groups need modification, a precursor of PCB
in the form of the parent alcohol can be used to form appropriate
ester-type linkages. These derivatives can be chemically linked to
a variety of macromolecules and molecular components including
amino acids, nucleotides, proteins and polypeptides, nucleic acids
(DNA, RNA, PNA), lipids, hormones and molecules which function as
ligands for receptors.
[0104] The application of biotin-avidin technology for the
detection and isolation of chemical and biological materials has
also been broadened by the use of binding molecules which are first
biotinylated and then allowed to selectively interact with the
target molecule to be isolated. Isolation of the target molecule or
cell is facilitated by the binding of the biotinylated
binding-complex to the streptavidin-containing column or
streptavidin-coated magnetic beads. Binding molecules include
antibodies which selectively bind to specific antigens, DNA probes
which selectively bind to specific DNA sequences and ligands which
selectively bind to specific receptors. This approach has been used
to isolate a wide variety of macromolecules and cells (Tables 2 and
3). However, such isolation methods require that the biotinylated
target be released from the bound streptavidin. Disruption of this
bond typically requires non-physiological conditions such as low pH
and high concentration of guanidinium-HCl which is usually damaging
for the target molecule or cell. Even after disruption of the
streptavidin-biotin interaction, the target or binding molecule
remains partially or completely biotinylated which can interfere
with later uses. Further, elution conditions are non-physiological
and can also be disruptive to the target molecule or cell. In
contrast, using photocleavable biotins substrate can be quickly and
easily cleaved from biotin with little to no effect on substrate
conformation or activity.
[0105] The use of PCB in any of the usual detection and
purification procedures, including those discussed above,
represents a significant savings of time, energy and ultimately
cost. In addition, a variety of derivatives of avidin and
streptavidin are commercially available which have been modified
through chemical or genetic means. These same derivatives can be
used with PCB. One example is ImmunoPure NeutrAvidin sold
commercially (Pierce Chemical; Rockford, Ill.). This protein is a
modified avidin derivative which has been deglycosylated and does
not contain the RYD domain that serves as a universal recognition
sequence for many cell receptors. Non-specific adsorption to other
proteins and cell surfaces is greatly reduced.
[0106] The major molecular elements of a photocleavable biotin
(PCB) are a photoreactive moiety and a biotinyl moiety which
constitutes the detectable moiety (FIG. 3). The photoreactive
moiety and the biotinyl moiety are linked together with a spacer
arm to form the PCB molecule. The photoreactive moiety contains a
five or six membered ring derivatized with functionalities
represented by X, Y and A-C(O)-G, wherein X allows linkage of PCB
to the biomolecular substrate. In the preferred embodiment, Y
represents a substitution pattern on a phenyl ring containing one
or more substituents such as nitro or alkoxy groups. The
functionality W represents the group that allows linkage of the
cross-linker moiety to the photoreactive moiety. The purpose of the
spacer arm is to increase the access of the biotin moiety for
effective interaction with streptavidin, and thus, increase the
binding efficiency. Typically these can be constructed using either
long alkyl chains or using multiple repeat units of caproyl
moieties linked via amide linkages.
[0107] Choice of photolabile group, spacer arm and the biotinyl
moiety depends on the target substrate including amino acids,
proteins, antibodies, nucleotides, DNA or RNA, lipids,
carbohydrates and cells to which the photocleavable biotin is to be
attached. It also depends on the exact conditions for photocleavage
and the desired interaction between the biotinyl moiety and
streptavidin, avidin or their derivatives. Some of the various
choices for the photolabile group and linker arms for PCB are shown
in FIG. 2.
[0108] Additional types of coupling agents include antibodies,
antibody fragments and antigens. Antibodies have the advantage that
they can bind to their respective antigen with great specificity.
Substrates which are antigens can be detected by their ability to
specifically bind to available antibodies or to antibodies which
can be easily created. Useful antibodies or antibody fragments may
be monoclonal or polyclonal and are preferable of the class IgG,
but may also be IgM, IgA; IgD or IgE. Other preferred detectable
moieties include nucleic acids. Short sequences of RNA or DNA or
oligonucleotides, preferably less than about thirty nucleotides in
length and as short as four to ten nucleotides, can be detected by
their ability to specifically hybridize with a complementary
nucleic acid and detected directly or indirectly using PCR which
greatly amplifies a specific sequence that is subsequently
detected. In a similar fashion, binding proteins and
receptor-ligand combinations are also useful as detectable
labels.
[0109] The second component of the bioreactive agent is the
photoreactive moiety. The photoreactive moiety is a chemical moiety
capable of forming one or more covalent bonds with a substrate
which can be cleaved with electromagnetic radiation. These bonds
may be formed with a chemical group on the substrate such as, for
example, an amine, phenol, imidazole, aldehyde, carboxylic acid or
thiol. The photoreactive agent is a substituted aromatic ring
containing at least one polyatomic group and, optionally, one or
more monoatomic groups. The aromatic ring is preferably a five or
six-membered ring. The substitutions comprise the polyatomic and
optional monoatomic groups. The polyatomic group imparts electron
channeling properties to attract or repel electrons to certain
locations within the chemical structure, thereby creating or
establishing the conditions to create the selectively cleavable
covalent bonds. Some monoatomic groups such as halides can adjust
the frequency or wavelength of the electromagnetic radiation which
will induce cleavage. As such, monoatomic groups fine tune the
cleavage event to sensitize conjugates to predetermined frequencies
or intensities of radiation.
[0110] One class of photoreactive moieties are 2-nitrobenzyl
derivatives. In their ground state, 2-nitrobenzyl-based agents and
conjugates have an intramolecular hydrogen bond between benzylic
hydrogen and the ortho nitro group (--CH . . . O.sub.2N) (B.
Brzezinski et al., J. Chem. Soc. Perkin. Trans. 2:2257-61, 1992).
Upon illumination with wavelengths of greater than 300 nm, these
chemical compounds transition to an excited state. Proton transfer
reaction from benzylic carbon to the oxygen in nitro group takes
place which is followed by electron rearrangement (FIG. 4). This
reaction results in the formation of a transient species called an
aci-nitro ion which is in a rapid equilibrium with a cyclic form.
In the cyclic intermediate, electron rearrangement and oxygen
transfer from nitrogen to benzylic carbon takes place resulting in
the formation of 2-nitroso derivatives and release of a substrate
which is a good leaving group (J. A McCray et al., Annu. Rev.
Biophys. Chem. 18:239-70, 1989).
[0111] Chemical synthesis of PCB NHS-ester involves three principal
steps: (1) Generation of the photoreactive moiety, for example,
5-methyl-2-nitroacetophenone. (2) Generation of a suitable amino
group and attachment to biotin containing spacer. (3) Generation of
hydroxyl groups and derivatization as N-hydroxysuccinimidyl
carbonate (NHS-ester). These steps are schematically represented in
FIG. 5.
[0112] Bioreactive agents can also be synthesized based on other
photoreactive moieties. Chemical syntheses of two other classes of
photocleavable moieties 3,5-dimethoxybenzyl and
2-nitrobenzenesulfenyl (FIG. 2) can be carried out using similar
synthesis strategies. These 2-nitrobenzyl groups all contain a
benzylic carbon-hydrogen bond ortho to a nitro group, which is
necessary for their photolability. In the developments of these
photolabile groups as protecting groups, difficulties were
encountered as the subsequent reactions of these carbonyl compounds
resulted in formation of coupled azo compounds, which act as
internal light filters (V.N.R. Pillai, Synthesis 1, 1980). These
complications were overcome in the present invention with the use
of .alpha.-substituted, o-nitrobenzyl compounds. Bioreactive agents
of the invention that form a detectable photocleavable conjugate
can, for example, be represented by the formula:
##STR00003##
wherein X is selected from the group consisting of a halogen,
N.sub.2, CH.sub.2-halogen, --N.dbd.C.dbd.O, --N.dbd.C.dbd.S,
--S--S--R, NC.sub.2H.sub.4, --NC.sub.4H.sub.2O.sub.2, --OH,
--NHNH.sub.2, --OP(OR.sub.3)N(R.sub.4)R.sub.5 and --OCO-G wherein G
is selected from the group consisting of a halogen, N.sub.3,
O-esters and N-amides; R.sub.1, R.sub.2, R.sub.3, R.sub.4 and
R.sub.5 are selected from the group consisting of hydrogen, alkyls,
substituted alkyls, aryls and substituted aryls, --CF.sub.3,
--NO.sub.2, --COOH and --COOR, and may be the same or different; A
is a divalent functional group selected from the group consisting
of --O--, --S-- and --NR.sub.1; Y comprises one or more polyatomic
groups which may be the same or different; V comprises one or more
optional monoatomic groups which may be the same or different; Q
comprises an optional spacer moiety; m1 and m2 are integers from
0-5 and may be the same or different; and D comprises a selectively
detectable moiety which is distinct from R.sub.1--R.sub.5. The
O-ester may be cyanomethyl, o and p nitrophenyl, 2,4-dinitrophenyl,
2,4,5-trichlorophenyl, pentachlorophenyl, pentafluorophenyl,
N-hydroxyphthalimidyl, N-hydroxysuccinimidyl,
1-hydroxypiperidinyl,5-chloro-8-hydroxy-quinolyl,
1-hydroxybenzotriazolyl,3,4-dihydro-4-oxobenzotriazin-3-yl (DHBT),
2,3-dihydro-2,5-diphenyl-3-oxo-thiophen-1,1-dioxide-4-yl (TDO),
1,2-benzisoxasolyl, 2-hydroxypyridyl or derivatives or combinations
thereof. The N-amide is an imidazolyl, benzimidazolyl,
benzisoxazolyl, 3,5-dioxo-4-methyl-1,2,4-oxadiezolidinyl or
derivatives or combinations thereof.
[0113] Polyatomic groups can be attached to the aromatic ring
include nitro groups (--NO.sub.2), sulfoxide groups such as
(--SO.sub.3), alkyl groups such as methyl (--CH.sub.3) and ethyl
groups (--CH.sub.2CH.sub.3), alkoxyl groups such as (--OCH.sub.3),
and derivatives and combinations thereof. Useful monoatomic groups
are halides such as chloride (--Cl), fluoride (--F), iodide (--I),
bromide (--Br), and hydrogen (--H). These groups may be placed in
any of the available positions around the ring. Selection and
placement of the polyatomic and monoatomic groups may influence the
wavelength of electromagnetic radiation required to induce cleavage
and the period of time that radiation must be applied to induce
efficient cleavage. The chemical moieties at R.sub.1, R.sub.2,
R.sub.3, R.sub.4 and/or R.sub.5 may also influence the
photoreaction.
[0114] It is sometimes useful to include in the agent a spacer
moiety bonded between the photoreactive moiety and the detectable
moiety. The presence of the spacer can be advantageous sterically
for substrate binding. The spacer moiety (Q) may comprise a
branched or straight chain hydrocarbon, a polymeric carbohydrate,
or a derivative or combination thereof. The preferred spacer moiety
is represented by the formula:
##STR00004##
[0115] wherein W and W' are each selected from the group consisting
of --C(O)--, --C(O)--NH--, --HN--C(O)--, --NH--, --O--, --S-- and
--CH.sub.2--, and may be the same or different; and n1 and n2 are
integers from 0-10 which can be the same or different and if either
n1 or n2 is zero, then W and W' are optional. Examples of the
chemical structure of bioreactive agents are depicted in FIG.
6.
[0116] Another embodiment of the invention is directed to
photocleavable conjugates comprising bioreactive agents
photocleavably coupled to substrates. Conjugates have the property
that they can be selectively cleaved with electromagnetic radiation
to release the substrate. Substrates are those chemicals,
macromolecules, cells and other substances which are or can be used
to detect and isolate targets. Substrates that are selectively
cleaved from conjugates may be modified by photocleavage, but still
functionally active, or may be released from the conjugate
completely unmodified by photocleavage. Substrates may be coupled
with agents, uncoupled and recoupled to new agents at will.
[0117] Useful substrates are any chemical, macromolecule or cell
that can be attached to a bioreactive agent. Examples of useful
substrates include proteins, peptides, amino acids, amino acid
analogs, nucleic acids, nucleosides, nucleotides, lipids, vesicles,
detergent micells, cells, virus particles, fatty acids,
saccharides, polysaccharides, inorganic molecules and metals.
Substrates may also comprise derivatives and combinations of these
substances such as fusion proteins, protein-carbohydrate complexes
and organo-metallic compounds. Substrates may also be
pharmaceutical agents such as cytokines, immune system modulators,
agents of the hematopoietic system, recombinant proteins,
chemotherapeutic agents, radio-isotopes, antigens, anti-neoplastic
agents, enzymes, PCR products, receptors, hormones, vaccines,
haptens, toxins, antibiotics, nascent proteins, synthetic
pharmaceuticals and derivatives and combinations thereof.
[0118] Substrates may be targets or part of the targets such as an
amino acid in the synthesis of nascent polypeptide chains wherein
substrates may be amino acid or amino acid derivative which becomes
incorporated into the growing peptide chain. Substrates may also be
nucleotides or nucleotide derivatives as precursors in the
synthesis of a nucleic acid. Constructs useful in creating
synthetic oligonucleotide conjugates may contain phosphoramidites
or derivatives of dATP, dCTP, dTTP and dGTP, and also ATP, CTP, UTP
and GTP. Resulting nucleic acid-conjugates can be used in PCR
technologies, antisense therapy, and prophylactic and diagnostic
applications. Substrates may be targets, for example, when it is
possible to specifically react the bioreactive agent with substrate
in a mixture such that the reaction creates the conjugate. Such
conjugates are useful when it is desirable to follow a target
through a biological or other type of system such as when
determining the half-life of a pharmaceutical.
[0119] Photocleavage of conjugates of the invention should
preferably not damage released substrate or impair substrate
activity. Proteins, nucleic acids and other protective groups used
in peptide and nucleic acid chemistry are known to be stable to
most wavelengths of radiation above 300 mL PCB carbamates, for
example, undergo photolysis upon illumination with long-wave UV
light (320-400 nm), resulting in release of the unaltered substrate
and carbon dioxide (FIG. 7). The yield and exposure time necessary
for release of substrate photo-release are strongly dependent on
the structure of photoreactive moiety. In the case of unsubstituted
2-nitrobenzyl PCB derivatives the yield of photolysis and recovery
of the substrate are significantly decreased by the formation of
side products which act as internal light filters and are capable
of reacting with amino groups of the substrate. In this case,
illumination times vary from about 1 minute to about 24 hours,
preferably less than 4 hours, more preferably less than two hours,
and even more preferably less than one hour, and yields are between
about 1% to about 95% (V.N.R. Pillai, Synthesis 1, 1980). In the
case of alpha-substituted 2-nitrobenzyl derivatives (methyl,
phenyl), there is a considerable increase in rate of photo-removal
as well as yield of the released substrate (J. E. Baldwin et al.,
Tetrahedron 46:6879, 1990; J. Nargeot et al., Proc. Natl. Acad.
Sci. USA 80:2395, 1983).
[0120] The choice of a particular bioreactive agent depends on
which molecular groups of the substrate are to be derivatized. For
example, reaction of photocleavable biotin NHS-ester with a protein
results in formation of a covalent bond with primary amino groups
such as at the .epsilon.-position of lysine residues or the
.alpha.-NH.sub.2 group at the N-terminal of a protein. Normally, a
number of lysine residues are exposed on the surface of a protein
and available for such reaction. Alternatively, several other
photocleavable biotins can be used which react with hydroxyl groups
(--OH) present in tyrosine, threonine and serine residues, carboxyl
groups (--COOH) present in aspartate and glutamate residues, and
sulfhydryl groups (--SH) present in cysteine residues (Table 4).
Thus, a wide variety of groups are available which are likely to be
on the surface of a target protein.
[0121] Attachment of photocleavable biotin to molecules which bind
proteins such as receptor ligands, hormones, antibodies, nucleic
acids, and proteins that bind glycoproteins can also be
accomplished because of the wide variety of reactive groups
available for photocleavable biotins. For example, photocleavable
biotin can be conveniently linked to antibodies which are directed
against a particular protein. Alternatively, photocleavable biotins
can be linked to DNA and RNA or to a variety of small molecules
including receptor ligands and hormones. The importance of
biotinylation of binding-complexes for isolation of proteins such
as membrane receptors and splicesomes has already been demonstrated
using conventional biotins or non-photocleavable biotins.
[0122] The choice of the detectable moiety depends on the
substrate, its environment and the desired method of detection and
isolation. For example, a substrate present in low concentrations
may require a sensitive method of detection such as fluorescent
spectroscopy thereby requiring a fluorescent moiety such as
coumarin. The wavelength of fluorescent emission can be selected by
the choice of detectable moiety so as not to interfere with any
natural fluorophores which may be present in the mixture. In cases
where rapid isolation of the substrate is desired, choice of the
detectable moiety may be determined by the availability of a
suitable coupling agent. For example, an antigen which serves as
the detectable moiety may be used if a suitable antibody is
available. Since the detectable moiety, the reactive group and the
photoreactive moieties are chemically separate in the bioreactive
agent, the properties of each can be adjusted to meet the multiple
requirements for detection and isolation of a particular
substrate.
[0123] Conjugates of the invention may be attached to a solid
support via the detectable moiety, the substrate or any other
chemical group of the structure. The solid support may comprise
constructs of glass, ceramic, plastic, metal or a combination of
these substances. Useful structures and constructs include plastic
structures such as microtiter plate wells or the surface of sticks,
paddles, beads or microbeads, alloy and inorganic surfaces such as
semiconductors, two and three dimensional hybridization and binding
chips, and magnetic beads, chromatography matrix materials and
combinations of these materials. Examples of the chemical structure
of conjugates of the invention include:
##STR00005##
[0124] wherein SUB comprises a substrate; R.sub.1 and R.sub.2 are
selected from the group consisting of hydrogen, alkyls, substituted
alkyls, aryls, substituted aryls, --CF.sub.3, --NO.sub.2, --COOH
and --COOR, and may be the same or different; A is a divalent
functional group selected from the group consisting of --O--, --S--
and --NR.sub.1; Y comprises one or more polyatomic groups which may
be the same or different; V comprises one or more optional
monoatomic groups which may be the same or different; Q comprises
an optional spacer moiety; m1 and m2 are integers between 1-5 which
can be the same or different; and D comprises a selectively
detectable moiety which is distinct from R.sub.1 and R.sub.2.
[0125] As discussed above, the polyatomic group may be one or more
nitro groups, alkyl groups, alkoxyl groups, or derivatives or
combinations thereof. The optional monoatomic group may be one or
more fluoro, chloro, bromo or iodo groups, or hydrogen. The
polyatomic and monoatomic groups and the chemical moieties at
R.sub.1 and R.sub.2 may effect the photocleavage reaction such as
the frequency of radiation that will initiate photocleavage or the
exposure time needed to execute a cleavage event. The spacer moiety
(Q) may be a branched or unbranched hydrocarbon or a polymeric
carbohydrate and is preferably represented by the formula:
##STR00006##
wherein W and W' are each selected from the group consisting of
--CO--, --O--NH--, --HN--CO--, --NH--, --O--, --S-- and
--CH.sub.2--, and may be the same or different; and n1 and n2 are
integers from 0-10 which can be the same or different and if either
n1 or n2 is zero, then W and W' are optional. Specific examples of
conjugates of the invention are depicted in FIG. 8.
[0126] Another embodiment of the invention is directed to
conjugates which are pharmaceutical compositions. Compositions must
be safe and nontoxic and can be administered to patients such as
humans and other mammals. Composition may be mixed with a
pharmaceutically acceptable carrier such as water, oils, lipids,
saccharides, polysaccharides, glycerols, collagens and combinations
thereof and administered to patients.
[0127] Pharmaceutical compositions with photo-releasable substrates
are useful for example, for delivery of pharmaceutical agents which
have short half-lives. Such agents cannot be administered through
current means without being subject to inactivation before having
an effect. Pharmaceutical agents in the form of conjugates,
covalently bound to bioreactive agents, are more stable than
isolated agents. After general administration of the composition to
the patient, the site to be treated is exposed to appropriate
radiation releasing substrate which produces an immediate positive
response in a patient. Uncoupling from the bioreactive agent at the
point of maximal biological effect is an advantage unavailable
using current administration or stabilization procedures. In an
analogous fashion, other areas of the patient's body may be
protected from the biological effect of the pharmaceutical agent.
Consequently, using these conjugates, site-directed and
site-specific delivery of a pharmaceutical agent is possible.
[0128] Another embodiment of the invention is directed to a method
for isolating targets from a heterologous mixture. Bioreactive
agents are contacted with the mixture to react with target forming
the conjugate. Alternatively, conjugates can be contacted with the
heterologous mixture to couple substrate within the conjugate to
one or more targets. Conjugates can be separated from the mixture
by any currently available techniques (e.g. Table 5).
TABLE-US-00005 TABLE 5 Affinity Techniques Using Avidin Material
Method of Separation Magnetic beads coated with Magnetic separation
Streptavidin Beads coated with Streptavidin Washing (e.g.
centrifugation) and elution Biotinylated Antibodies
Immunoprecipitation Cross-linked-bisacrylamide/azolactone Column
Chromatography copolymers with avidin Agarose coated with
Streptavidin Column Chromatography
[0129] Procedures such as chemical or physical separation of
components of the mixture, electrophoresis, electroelution,
sedimentation, centrifugation, filtration, magnetic separation,
chemical extraction, affinity separation methods such as affinity
chromatography or another chromatographic procedure such as
ion-exchange, gradient separation, HPLC or FPLC, and combinations
of these techniques are well-known and allow for a rapid isolation
with a high efficiency of recovery (e.g. M. Wilchek et al., Methods
Enzymol. 184, 1990; M. Wilchek et al., Anal. Biochem. 171:1, 1988).
After separation or isolation, targets can be easily quantitated
using available methods such as optical absorbance or transmission
(e.g. nucleic acid, proteins, lipids) or the Bradford (M. Bradford,
Anal. Biochem. 72:248, 1976) or Lowry (0. Lowry et al., J. Biol.
Chem. 193:265, 1951) assays (e.g. proteins), both of which are
commercially available. After separation, coupled conjugates are
treated with electromagnetic radiation to release substrate. The
substrate targets can than be separated from the released
bioreactive agent, if desired, to obtain substantially or
completely pure targets.
[0130] Targets which can be detected and isolated in a highly
purified form by this method include nearly any chemical, molecule
or macromolecule including immune system modulators, agents of the
hematopoietic system, cytokines, proteins, hormones, gene products,
antigens, cells including fetal and stem cells, toxins, bacteria,
membrane vesicles, virus particles, and combinations thereof.
Detection and isolation are determined by the ability of the
bioreactive agent to bind substrate. For example, nucleic acids can
be base-paired to complementary nucleic acids, to nucleic acid
binding proteins or to chemical moieties which react specifically
with chemical moieties found on nucleic acids. Proteins can be
bound with monoclonal or polyclonal antibodies or antibody
fragments specific to those proteins, or chemical moieties which
react specifically with chemical moieties found on the proteins of
interest. Substrates may be, for example, precursors of targets
such as one or more of the naturally or non-naturally occurring
amino acids wherein the target is a nascent protein, or one or more
ribonucleotides, deoxyribonucleotide or primers when the target is
a nucleic acid. Precursor can be incorporated into target molecules
by, for example, in vivo or in vitro replication, transcription or
translation. Target may be a protein or protein-containing complex,
nucleic acid, gene sequence or PCR product. Substrates may also be
receptors which bind to or otherwise associate with ligands
specific for the receptor molecules. Receptors which can be
isolated include cytokines wherein the target is a cytokine
receptor and antigens wherein the target is an antibody. Preferred
conjugates for the detection and isolation of a target from a
heterologous mixture are photocleavable biotins linked to
antibodies (polyclonal, monoclonal fragments), photocleavable
coumarins linked to antibodies, photocleavable dansyls linked to
lipids and derivatives and modifications thereof.
[0131] The heterologous mixture which contains target may be a
biological sample, any proteinaceous composition such as a cellular
or cell-free extract, nucleic acid containing compositions, a
biomass containing, for example, vegetative or microbial material,
a cell culture of primary or immortalized cells, lipid vesicles or
even animals. Animals may be used to detect targets which may be
present in the body or parts of the body or, alternatively, to
collect and isolate targets such as macromolecules or cells from
animal models. Substrate can also be proteins, peptides, amino
acids, amino acid analogs, nucleosides, nucleotides, lipids,
vesicles, detergent micells, fatty acids, saccharides,
polysaccharides, inorganic molecules, metals and derivatives and
combinations thereof.
[0132] In an application of this method, the substrate may be an
integral component of the target such as a nucleotide in the
detection and isolation of nascent nucleic acids or an amino acid
in the detection and isolation of nascent proteins. Substrate is
incorporated into target by chemical or enzymatic techniques and
detected and isolated by the presence of the detectable moiety.
Briefly, conjugates are contacted with reagents in a heterologous
mixture such as, for example, in a replication, transcription,
translation or coupled transcription/translation system. Substrates
are incorporated into targets through the action of components in
the system such as enzymes, precursor molecules and other reagents
of the system. Conjugate coupled targets are separated from the
mixture and treated with electromagnetic radiation to release the
target which is then isolated.
[0133] Conjugates can be contacted with a heterologous mixture by
incubation as in, for example, the enzymatic incorporation of a
macromolecular precursor into a nascent macromolecule which may be
either in vivo or in vitro. Nucleic acid polymerases will
incorporate precursor nucleotides or nucleic acid primers into
nucleic acids. In vitro incubations in cell-free reaction mixture
are typically performed at a temperature of between about 4.degree.
C. to about 45.degree. C., preferably at between about 12.degree.
C. to about 37.degree. C., and more preferably at about room
temperature. Incubation of conjugates into nascent macromolecules
may be complete in about 5 minutes, about 15 minutes, or about one
hour depending on the incubation conditions, or may require two,
three or more hours to complete. When the heterologous mixture is
an animal or an animal model in vivo incubations are generally
performed at body temperature and may require hours or days for
conjugates to distribute to areas of the animal's body which may be
remote from the site of introduction, for conjugates to react with
targets and for conjugates coupled with targets to be
collected.
[0134] One of the preferred embodiments of the invention relates to
the detection or isolation of protein using photocleavable biotic.
In one application of this embodiment, PCB is reacted with a
protein through the formation of covalent bonds with specific
chemicals groups of the protein forming a conjugate. The protein
may be either the target to be isolated or detected or a probe for
the target protein such as an antibody. The target protein can then
be isolated using streptavidin affinity methodology.
[0135] Another application of this embodiment is directed to the
use of photocleavable biotin to isolate nascent proteins that can
be created from in vitro or in vivo protein synthesis. Basically,
photocleavable biotins are synthesized and linked to amino acids
(PCB-amino acids) containing special blocking groups. These
conjugates are charged to tRNA molecules and incorporated into
peptides and proteins using a translation or coupled
transcription/translation system. PCB-amino acids of the invention
have the property that once illuminated with light, a photocleavage
occurs that produces a native amino acid plus the free biotin
derivative. Such proteins can be isolated in a structurally and/or
functionally unaltered form.
[0136] The detailed procedure for the production of photocleavable
biotin amino acids and their incorporation into the nascent
proteins involves a few basic steps. First, photocleavable biotin
is synthesized and linked to an amino acid with an appropriate
blocking group. These PCB-amino acid conjugates are charged to tRNA
molecules and subsequently incorporated into nascent proteins in an
in vivo or in vitro translation system. Alternatively, a tRNA
molecule is first charged enzymatically with an amino acid such as
lysine which is then coupled to a reactive PCB. Nascent proteins
are separated and isolated from the other components of synthesis
using immobilized streptavidin. Photocleavage of PCB-streptavidin
complex from the nascent protein generates a pure and native,
nascent protein.
[0137] PCB is attached to an amino acid using, for example, the
side-chain groups such as an amino group (lysine), aliphatic and
phenolic hydroxyl groups (serine, threonine and tyrosine),
sulfhydryl group (cysteines) and carboxylate group (aspartic and
glutamic acids) (FIG. 9). Synthesis can be achieved by direct
condensations with appropriately protected parent amino acids. For
example, lysine side chain amino group can be modified with PCB by
modification of the .epsilon.-amino group. The synthesis of for
example, PCB-methionine involves primarily .alpha.-amino group
modification. PCB-methionine can be charged to an initiator tRNA
which can participate in protein synthesis only at initiation sites
which results in single PCB incorporation per copy of the nascent
protein.
[0138] One method for incorporation of a photocleavable biotin
amino acid into a nascent protein involves misaminoacylation of
tRNA. Normally, a species of tRNA is charged by a single, cognate
native amino acid. This selective charging, termed here enzymatic
aminoacylation, is accomplished by enzymes called aminoacyl-tRNA
synthetases and requires that the amino acid to be charged to a
tRNA molecule be structurally similar to a native amino acid.
Chemical misaminoacylation can be used to charge a tRNA with a
non-native amino acids such as photocleavable amino acids. The
specific steps in chemical misaminoacylation of tRNAs are depicted
in FIG. 10.
[0139] As shown, tRNA molecules are first truncated to remove the
3'-terminal residues by successive treatments with periodate,
lysine (pH 8.0) and alkaline phosphate (Neu et al., J. Biol. Chem.
239:2927-34, 1964). Alternatively, truncation can be performed by
genetic manipulation, whereby a truncated gene coding for the tRNA
molecule is constructed and transcribed to produce truncated tRNA
molecules (Sampson et al., Proc. Natl. Acad. Sci. USA 85:1033,
1988). Second, protected acylated dinucleotides, pdCpA, are
synthesized (Hudson, J. Org. Chem. 53:617, 1988; E. Happ, J. Org.
Chem. 52:5387, 1987). PCB-amino acids blocked appropriately at
their side chains and/or at .alpha.-amino groups, using standard
protecting groups like Fmoc, are prepared and coupled with the
synthetic dinucleotide in the presence of carboxy group activating
reagents. Subsequent deprotection of Fmoc groups yields
aminoacylated dinucleotide.
[0140] Third, the photocleavable biotin amino acid is ligated to
the truncated tRNA through the deprotected dinucleotide. The bond
formed by this process is different from that resulting from tRNA
activation by an aminoacyl-tRNA synthetase, however, the ultimate
product is the same. T4 RNA ligase does not recognize the O-acyl
substituent, and is thus insensitive to the nature of the attached
amino acid (FIG. 10). Misaminoacylation of a variety of non-native
amino acids can be easily performed. The process is highly
sensitive and specific for the structures of the tRNA and the amino
acid.
[0141] Aminoacylated tRNA linked to a photocleavable biotin amino
acid can also be created by employing a conventional aminoacyl
synthetase to aminoacylate a tRNA with a native amino acid or by
employing specialized chemical reactions which specifically modify
the native amino acid linked to the tRNA to produce a
photocleavable biotin aminoacyl-tRNA derivative. These reactions
are referred to as post-aminoacylation modifications. Such post
aminoacylation modifications do not fall under the method of
misaminoacylation, since the tRNA is first aminoacylated with its
cognate described amino acid.
[0142] In contrast to chemical aminoacylation, the use of
post-aminoacylation modifications to incorporate photocleavable
biotin non-native amino acids into nascent proteins is very useful
since it avoids many of the steps including in misaminoacylation.
Furthermore, many of the photocleavable biotin derivatives can be
prepared which have reactive groups reacting specifically with
desired side chain of amino acids. For example, postaminoacylation
modification of lysine-tRNA.sup.Lys, an N-hydroxysuccinimide
derivative of PCB can prepared that would react with easily
accessible primary .epsilon.-amino and minimize reactions occurring
with other nucleophilic groups on the tRNA or .alpha.-amino groups
of the amino acylated native amino acid. These other non-specific
modifications can alter the structure of the tRNA structure and
severely compromise its participation in protein synthesis.
Incomplete chain formation could also occur when the .alpha.-amino
group of the amino acid is modified. Post-aminoacylation
modifications to incorporate lysine-biotin non-native amino acids
into nascent proteins has been demonstrated (tRNA.sup.nscend.TM.;
Promega; Madison, Wis.) used for the detection of nascent protein
containing biotin using Western Blots followed by enzymatic assays
for biotin (T. V. Kurzchalia et al., Eur. J. Biochem. 172:663-68,
1988). However, these biotin derivatives are not photocleavable
which, in the case of NHS-derivatives of PCB, allows the biotin
linkage to the lysine to be photochemically cleaved.
[0143] PCB-amino acids can also be incorporated into polypeptide by
means of solid-support peptide synthesis. First, PCB-amino acids
are derivatized using base labile fluorenylmethyloxy carbonyl
(Fmoc) group for the protection of .alpha.-amino function and acid
labile t-butyl derivatives for protection of reactive side chains.
Synthesis is carried out on a polyamide-type resin. Amino acids are
activated for coupling as symmetrical anhydrides or
pentafluorophenyl esters (E. Atherton et al., Solid Phase Peptide
Synthesis, IRL Press, Oxford, 1989). Second, amino acids and PCB
are coupled and the PCB-amino acid integrated into the polypeptide
chain. Side chain PCB-derivatives, like .epsilon.-amino-Lys, side
chain PCB-amino acid esters of Glu and Asp, esters of Ser, Thr and
Tyr, are used for incorporation at any site of the polypeptide.
PCB-amino acids may also be incorporated in a site-specific manner
into the chain at either predetermined positions or at the
N-terminus of the chain using, for example, PCB-derivatized
methionine attached to the initiator tRNA.
[0144] A wide range of polypeptides can be formed from PCB-amino
acids cytokines and recombinant proteins both eukaryotic and
prokaryotic (e.g. .alpha.-, .beta.- or .gamma.-interferons;
interleukin-1, -2, -3, etc.; epidermal fibroblastic, stem cell and
other types of growth factors), and hormones such as the
adrenocorticotropic hormones (ACTHs), insulin, the parathyroid
hormone (bPTH), the transforming growth factor .beta. (TGF-.beta.)
and the gonadotropin releasing hormone (GnRH) (M. Wilchek et al.,
Methods Enzymol. 184:243, 1990; F. M. Finii et al., Methods
Enzymol. 184:244, 1990; W. Newman et al., Methods Enzymol. 184:275,
1990; E. Hazum, Methods Enzymol. 184:285, 1990). These hormones
retain their binding specificity for the hormone receptor. One
example is the GnRH hormone where a biotin was attached to the
epsilon amino group Lys-6 through reaction of a d-biotin
p-nitophenyl ester. This biotinylated hormone can be used for
isolation of the GnRH receptor using avidin coated columns.
[0145] After incorporation or attachment of PCB into a protein,
protein-complex or other amino acid-containing target, the target
is isolated using a simple four step procedure (FIG. 11). First, a
bioreactive agent (PCB) is synthesized. Second, a substrate is
coupled to the bioreactive agent forming a conjugate. Third, target
is separated from other materials in the mixture through the
selective interaction of the photocleavable biotin with avidin,
streptavidin or their derivatives. Captured targets may be
immobilized on a solid support such as magnetic beads, affinity
column packing materials or filters which facilitates removal of
contaminants. Finally, the photocleavable biotin is detached from
the target by illumination of a wavelength which causes the
photocleavable biotin covalent linkage to be broken. Targets are
dissolved or suspended in solution at a desired concentration. In
those situations wherein conjugate coupled targets are not attached
to solid supports, release of targets can be followed by another
magnetic capture to remove magnetic particles now containing
avidin/streptavidin bound biotin moiety released form the
photocleavage of PCB. Thus, a completely unaltered protein is
released in any solution chosen, in a purified form and at nearly
any concentration desired.
[0146] Another example for the use of PCB is where the conjugate
comprises a PCB-coupled antibody. The use of photocleavable biotin
provides a means for recovering target molecule and the antibody
for subsequent use. Release of a protein from binding complex can
be performed subsequent to release of the binding complex from the
immobilized streptavidin. This is an advantage since it enables the
release to be performed under well controlled conditions. For
example, elution of a target protein from an affinity column often
requires changes in buffer and/or use of a competitive agent such
as an epitope which competes for an antibody binding site. This can
require long exposure of the protein to damaging conditions or the
need for increased amounts of the competitive agent which can be
prohibitively expensive. In contrast, once the protein complex is
removed from the immobilized streptavidin by photocleavage, the
complex can be separated more conveniently. In the case where
antibodies are used as the substrate, an additional advantage of
the invention is that the antibody can also be recovered in an
unaltered and purified form.
[0147] A simple scheme for using a PCB-antibody is shown in FIGS.
12A and 12B. Target protein/antibody conjugated to photocleavable
biotin is then immobilized with streptavidin. Target
protein/antibody is decoupled by illumination with light and the
target protein/antibody released. Target protein is separated from
antibody using, for example, increased or decreased salt (NaCl,
KCl) concentrations and recovered. Alternatively, as shown in FIG.
12B, the target is removed from the antibody by using an
epitope-PCB conjugate which competes for the antibody binding site.
The antibody-epitope-PCB conjugate is then isolated from the target
by immobilization with streptavidin. The antibody-epitope complex
is then released by photocleaving the biotin-epitope complex. A
wide variety of other molecules that interact with proteins can
also be utilized in conjunction with PCB for protein isolation, as
shown in Table 2, including polypeptides, protein complexes and
small ligands.
[0148] PCB derivatives attached or incorporated into antibodies or
other macromolecules such as DNA which serve as hybridization
probes can also be used advantageously for sequential multiple
detection of targets. As in the case of conventional assays for
target based on streptavidin-biotin interactions, the presence of
the target is signaled by a streptavidin-enzyme complex which binds
to the biotinylated probe and produces an amplified signal by
converting a substrate into a product which is easily detectable
due to a distinctive physical property such as color or
luminescence. However, in contrast to conventional biotins, the PCB
derivatives can be completely removed allowing for separation
removal of the streptavidin-enzyme complex and subsequent addition
of new probes and streptavidin complexes for the detection of
additional target molecules. A similar advantage exists for
cytochemical labeling based on streptavidin-biotin interaction. In
this case, the label can be completely removed by light, allowing
for additional specific cytochemical labels to be used.
[0149] In another application of the preferred embodiment,
photocleavable biotin or a PCB derivative can be incorporated into
a DNA (deoxyribonucleic acid), RNA (ribonucleic acid) or PNA
(polynucleic amide; P. E. Nielsen et al., Sci. 254:1497-1500, 1991)
molecule produced by, for example, chemical synthesis, PCR, nick
translation or DNA or RNA polymerases (Table 6). Target DNA or RNA
can then be isolated by using streptavidin affinity methods similar
to methods discussed above. Isolation is accomplished, for example,
using commercially available magnetic beads coated with
streptavidin. Beads will bind tightly to all DNA and RNA containing
the PCB derivative, whereas all other molecules are washed away.
The photocleavable biotin is then removed from the target nucleic
acid by illumination.
[0150] The incorporation of PCB into nucleic acids such as DNA and
RNA involves the synthesis and use of compounds that are formed
from the derivatization of nucleotides with photocleavable biotins.
Examples of the nucleotides modified using photocleavable biotin
are shown FIG. 13A.
[0151] Non-specific incorporation of PCB moieties into nucleic
acids is achieved, for example, using bisulfite catalyzed cytosine
transamination and subsequent reaction with PCB-NHS ester or
PCB-NH--NH.sub.2(PCB-hydrazide). Alternatively, 5'-phosphate can be
converted into phosphoramidite by reaction with water soluble
carbodiimide, imidazole and diamine and the resulting
phosphoramidite reacted with PCB-NHS ester.
[0152] Numerous methods have been developed to introduce an amino
group on 5'-end of protected oligonucleotides during solid support
synthesis. These include carbonyl diimidazole mediated modification
of 5'-OH with diamine and introduction of aliphatic amine moiety on
5'- or 3'-end during synthesis using special phosphoramidites.
Bifunctional phosphorarnidites have also been developed that allow
for incorporation of reactive amino group into multiple sites in
synthetic oligonucleotides. These methods produce oligonucleotide
products bearing aliphatic amino groups, which can be easily
converted into PCB-carbamates by reaction with respective PCB-NHS
esters. In an analogous manner as conventional biotin, PCB moieties
can be directly incorporated into oligonucleotides during solid
support synthesis using respective PCB-phosphoramidites.
[0153] Synthetic oligonucleotides of predetermined or random
sequences have a variety of uses as, for example, primers,
hybridization probes and antisense sequences. The synthesis of DNA
and RNA oligonucleotides utilizes phosphoramidite chemistry and is
routinely performed on an automated synthesizer with the growing
nucleic acid chains attached to a solid support such as CPG
(controlled pore glass). In this manner, phosphoramidites and other
reagents can be added in excess and removed by filtration. The
synthesis cycle comprises four reactions. First, acid labile trityl
groups are removed from the 5'-OH groups. Second, phosphoramidites
are coupled to the 5'-OH. Third, unreacted 5'-OH groups are
protected by capping with acetyl groups. Finally, internucleotide
linkage is converted from phosphite to phosphotriester by
oxidation. This cycle is repeated until the desired sequence is
obtained after which, oligonucleotide is cleaved from solid support
and purified using, for example, gel electrophoresis and HPLC.
[0154] Coupling efficiency for each step is preferably as high as
possible such as greater than 90% or about 97-99%. Unreacted
molecules are eliminated at each step by capping with acetyl groups
preventing the formation of undesired sequences. Crude
oligonucleotide contains besides full length sequence numerous
shorter sequences also called the failure sequences. Purification
of such a complex mixture is difficult especially when it comes to
isolation of full-length sequence from slightly shorter failure
sequences (e.g. n-1; n-2). This problem becomes even more difficult
when synthesizing long sequences of DNA or RNA where coupling
efficiencies are lower and the number of failure sequences higher.
5'-PCB-phosphoramidites can be used to selectively labile
full-length oligonucleotides at their 5'-end during solid-support
synthesis on automated nucleic acid synthesizers.
[0155] PCB-phosphoramidites can contain the PCB moiety linked to a
phosphoramidite functionality through a spacer arm allowing for
efficient binding to avidin. This reagent selectively reacts with
the 5'-OH group on a sugar ring and can be used on automated
synthesizers. Biotinylated sequences can also be prepared using
standard synthesis cycles with coupling efficiency being monitored
by trityl analysis. Typical coupling efficiencies are between about
90-95%. In addition, photocleavage of 5'-PCB nucleic acid results
in formation of 5'-phosphorylated sequences. 5'-phosphorylated
oligonucleotides are required for most applications in molecular
biology.
[0156] Alternatively, PCB moieties can also be incorporated during
synthesis into oligonucleotides at any position including the
3'-terminus, and into multiple sites using PCB-phosphoramidites
utilizing, for example, bifunctional non-nucleosidic backbones.
Enzymes including the Taq DNA polymerase used in PCR reactions, and
other DNA and RNA polymerases are capable of incorporating
biotinylated nucleotides into nucleic acids. PCR technology
comprises the process of amplifying one or more specific nucleic
acid sequences in a nucleic acid using primers and agents for
enzymatic polymerization followed by detection of the now amplified
sequence (R. K Saiki et al., Sci. 230:1350, 1985; T. J. White et
al., Trends Gent. 5:185, 1989). The basic techniques are described
in U.S. Pat. No. 4,683,195, which is specifically incorporated by
reference, and variations thereof described in U.S. Pat. Nos.
5,043,272, 5,057,410 and 5,106,727, which are also specifically
incorporated by reference. Several examples exist where
biotinylated nucleotides have been efficiently incorporated during
the PCR applications. These studies demonstrate that PCR carried
out in the presence of PCB-nucleotides results in a large
amplification of target DNA fragment and simultaneous labeling with
PCB. The primers required for PCR reaction may also be labeled.
[0157] Table 6 lists different enzymatic methods that would allow
incorporation of PCB-nucleotides to generate labeled probes for
many applications like in situ hybridization and PCR.
TABLE-US-00006 TABLE 6 Enzymatic Methods for Incorporation of
PCB-Nucleotides Method Substrate Enzyme Remarks Nick Translation
DNA or E. coli DNA pol I Most popular RNA method Replacement double
T4 DNA High specific synthesis stranded Polymerase incorporation in
using T4 DNA DNA dsDNA polymerase Reverse RNA Molony murine
Preparation of transcriptase leukemia virus long cDNA copies
reverse of RNA transcriptase 3'-Terminal ds DNA terminal
3'-hydroxyl labeling deoxynucleotidyl terminal labeling transferase
of dsDNA RNA labeling RNA SP6 Polymerase T3 RNA is the T3 or T7 RNA
most efficient for Polymerase RNA labeling Post-transcription RNA
SP6, T3 or T7 Pol Incorporate labeling allylamine-UTP first
followed by its modification by PCB 3'-labeling of RNA T4 RNA
ligase Based on ADP RNA (including derivatives of PCB. tRNAs
etc)
[0158] Biotin-avidin technology is currently used extensively in
the field of molecular biology and biomedicine as a means for
efficiently isolating the products of DNA and RNA synthesis as well
as for detection of specific sequences in nucleic acids. Isolation
normally involves the attachment or incorporation of biotin into
the DNA or RNA followed by separation through the interaction of
the biotin with streptavidin. For example, this methodology is used
widely for the isolation of DNA, which is the product of the
polymerase chain reaction. Detection typically involves the
preparation of biotin labeled nucleic acid probes. These probes
have found wide-spread application in gene structure and gene
function studies, the diagnosis of human, animal and plant
pathogens, and the detection of human genetic abnormalities.
[0159] However, conventional methodologies are limited by the
difficulty of detaching or releasing biotin from the nucleic acid.
In particular, it is highly desirable to obtain unaltered DNA or
RNA after it is separated by the biotin-avidin interaction. For
example, the presence of biotin on the nascent DNA can interfere
with its subsequent utilization in cloning or hybridization
analysis. In addition, the inability to remove biotin from a
biotinylated nucleic acid probe after an enzyme linked assay
prevents additional hybridization assays from being performed on
the same sample.
[0160] The utilization of photocleavable biotins in the isolation
or detection of nucleic acids eliminates many of the difficulties
listed above by providing a rapid and effective method of removing
the biotin in a single step. In the case of biotin-avidin based
isolation of DNA this also accomplishes the step of releasing the
DNA from the immobilized avidin.
[0161] In a preferred embodiment of this invention, the isolation
of nucleic acids is based on three basic steps. First, a
photocleavable biotin derivative is attached to a nucleic acid
molecule by enzymatic or chemical means or, alternatively, by
incorporation of a photocleavable biotin nucleotide into a nucleic
acid by enzymatic or chemical means. The choice of a particular
photocleavable biotin depends on which molecular groups are to be
derivatized on the nucleic acid. For example, attachment of
photocleavable biotin to a nucleic acid can be accomplished by
forming a covalent bond with the aromatic amine, sugar hydroxyls or
phosphate groups (Table 4). PCB can also be incorporated into
oligonucleotides through chemical or enzymatic means. Next, the
nucleic acid molecule is separated through the selective
interaction of the photocleavable biotin with avidin, streptavidin
or their derivatives which can be immobilized on a material such as
magnetic beads, affinity column packing materials or filters.
Methods for the separation of nucleic acids from other molecules in
a complex mixture using photocleavable biotin are well-established
and similar to the more conventional methods utilizing
non-cleavable biotin. This typically involves an affinity technique
based on streptavidin-biotin interaction whereby the nucleic acid
containing biotin is immobilized due to its interaction with
streptavidin. These techniques include, as shown in Table 5,
streptavidin-coated magnetic beads, streptavidin-sepharose columns
and streptavidin conjugated filters, all of which are commercially
available. For example, nucleic acid molecules containing PCB
either through attachment or incorporation are isolated using
streptavidin-coated magnetic beads. The pool of unbound
biomolecules is then washed to remove other reactants, buffer and
salts. Finally, the photocleavable biotin is detached from the
nucleic acid by illumination at a wavelength which causes the
photocleavable biotin covalent linkage to be broken. The
bioreactive agent can be removed leaving a substantially or
completely pure nucleic acid.
[0162] Another aspect of the invention is directed to the use of
photocleavable conjugates in conjunction with PCR amplification.
Methods for the isolation of a PCR product may use one or more
oligonucleotide primers as substrates. The nucleic acid sequence of
the target is PCR amplified using the conjugated primers. Covalent
bonds between the primer and the bioreactive agent are selectively
cleavable with electromagnetic radiation to release the amplified
sequences. Nucleotide sequences which can be selectively amplified
by this method includes nucleotide sequences found in biological
samples, bacterial DNA and eukaryotic DNA. In contrast to
conventional biotins, PCB offers an effective method to completely
remove biotinylated DNA product of the polymerase chain reaction
and provides a means to simultaneously release the PCR product from
the avidin or streptavidin binding medium and remove the
biotinylation in a single step.
[0163] Other advantages over conventional biotins include the
elimination of the need for special reagents or buffers. After
photocleavage of biotin from the PCR product, the resulting DNA is
suitable for cloning and other common usages in molecular biology.
After photocleavage of biotin from the PCR product, it can be
accurately analyzed with standard analytical methods such as gel
electrophoresis. Hybridization probes containing PCB can be
sterilized, wherein the biotin is completely removed so that the
target DNA can be reprobed using a second biotinylated probe. The
PCB incorporated into DNA retains the high binding affinity to
avidin unlike the several derivatives of biotin where properties of
biotin are attenuated for the purposes of easy release (e.g.
iminobiotin).
[0164] Two methods for the isolation of PCR products using PCB are
represented in FIGS. 14A and 14B. In both methods, the source of
the initial pool of cells from which the target DNA is to be
amplified can be from a wide variety of sources including
peripheral blood and biopsy tissues. After cell lysis, the crude
extract of total genome is subjected to the PCR.
[0165] In method A (FIG. 14A), DNA primers are synthesized from the
flanking sequences of the target DNA with photocleavable biotin
incorporation at the 5' ends. For this purpose, a
PCB-phosphoramidite can be introduced directly at the 5' end of the
oligonucleotide primer during DNA synthesis. A set of PCR cycles is
carried out using normal dNTPs. This procedure results in PCR
product where the photocleavable biotin is present on the 5' end of
each complementary strand. PCR products are separated from the
mixture containing other components of the PCR mixture including
nucleotides, enzymes, buffers by using streptavidin such as present
on coated magnetic beads (e.g. Dynabeads M-280 Streptavidin) which
bind the photocleavable biotin present at the 5' end of the DNA. A
typical procedure to be followed is to mix 40 .mu.l of washed
Dynabeads M-280 Streptavidin with 40 .mu.l of the PCR mixture and
to incubate for 15 minutes at room temperature. The Dynabeads are
then collected using magnetic means such as the DYNAL Magnetic
Particle Concentrators. Residual primers will be bound to the
streptavidin-binding material due to the presence of photocleavable
biotin at its 5' end. A small spin column like NAP-5 (Pharmacia
Biotech; Piscataway, N.J.) can be used to remove smaller molecules
and primers before streptavidin-magnetic bead capture is carried
out. The immobilized PCR product, now bound to streptavidin, is
photolyzed to release biotin from the DNA and the unmodified PCR
product recovered.
[0166] In method B (FIG. 14B), the problem of primers contamination
is eliminated. DNA primers are synthesized for the flanking
sequences of the target DNA without 5' biotinylation using
conventional oligonucleotide synthesis. Normal dNTPs along with a
small pool of PCB-dUTP are introduced into the PCR reactions. For
convenience, the dNTPs and PCB-dUTP can be premixed into aliquots
to be used in conjunction with PCR reactions. The PCR products are
separated from the mixture containing other components of the PCR
mixture including nucleotides, enzymes, buffers and primers by
using streptavidin such as present on coated magnetic beads (e.g.
Dynabeads M-280 Streptavidin).
[0167] The immobilized PCR product, now bound to streptavidin, is
photolyzed to release biotin from the DNA and recover unmodified
PCR product. Method A may sometimes be preferable when the
immobilized DNA which is bound to streptavidin is to be assayed
using a biotinylated probe. In this case the entire complex could
be released after assay by photocleavage followed by an addition
separation step, eliminating the biotinylated probe and leaving the
PCR product free for further use such as for cloning.
Alternatively, Method B may be preferable if release of a
primer-free product is required without an intermediate assay. This
would also produce a higher recovery since there are more PCB
molecules per molecule of DNA.
[0168] PCR is also widely used in the detection of a variety of
diseases related markers. Table 7 illustrates the various uses of
PCR for detection of diseases and disorders and the potential uses
of PCB-incorporated PCR in such diagnostic and forensic
applications.
TABLE-US-00007 TABLE 7 PCR use for detection of disease related
DNA/RNA Disease/ Assay of Virus/Bacteria Primer the PCR product
HTLV-I various targets including Liquid hybridization/Spot tax, gag
or env blot HIV targeted at the conserved Oligomer Hybridization
regions of the virus Hepatitis-B pol gene Southern hybridization
Papillomaviruses Dot-blot Restriction enzyme analysis
Cytomegalovirus Hybridization (herpes virus group) Enterovirus 100%
conserved regions RT-PCR followed by RNA hybridization
Gastroenteritis Reverse hybridization Cholera
[0169] In a preferred embodiment of the invention, the methodology,
based on PCB, for isolation and detection of PCR products can be
applied to diagnostic assays of a variety of diseases, the
detection of mutations as well as to the identification of unique
DNA sequences. For example, serological assays such as Western
blots and immunofluorescence and radioimmunoprecipitation assays
provide a rapid and sensitive procedure to screen for the presence
of antibodies to HIV-1. Further, in current PCR-based analysis,
highly conserved regions of the viral genomes are targeted for
amplification and involve hybridization using .sup.32P-labeled
oligomer probes in solution to one strand of an amplified product.
These tests can be used only for the direct detection of the virus.
A useful assay for the detection of HIV would detect not only
active virus, but also the presence of latent virus which has not
yet expressed its genome, but is still present in cells. This would
allow determination of both latent and actively replicating virus.
This would be particularly useful in newborns where maternal
antibodies can interfere with serological tests.
[0170] In another preferred embodiment, conjugates may be used to
efficiently create genomic or cDNA libraries. Construction of a
PCR-directed cDNA library from total RNA may provide the only
methodological approach to analyze cell-specific gene expression
where the amount of biological tissue is severely restricted. This
approach is particularly applicable where a specific stimulus
results in the modification or differentiation of a small number of
cells within a population.
[0171] Current schemes for the construction of, for example, cDNA
libraries require the isolation of cellular RNA and usually further
purification of mRNA from the more abundant rRNA and tRNA
components. The major advantage in PCR based methods is that mRNA
purification is not necessary. This is particularly advantageous
where biological material is limited wherein efficient mRNA
purification would be impossible. The general strategy is
illustrated schematically in FIG. 15.
[0172] High quality, intact RNA is prepared using guanidinium
hydrochloride procedure (S. J. Gurr et al., PCR: A Practical
Approach, Oxford University Press, New York, 1991). First strand
cDNA is synthesized using AMV reverse transcriptase in the presence
of PCB-dCTP in 1:1 ratio with dCTP (final concentration of both
should equal that of other dNTPs). This is followed by removal of
oligo-dT primers which can be carried out in a rapid and
quantitative manner using magnetic capture followed by
illumination. Current methods use CTAB precipitation which results
in loss of valuable cDNA:RNA hybrids, which is followed by
homopolymer tailing using oligo-dG (A. Otsuka, Gene 13:339, 1981).
After homopolymer tailing, the RNA is hydrolyzed which subjects
cDNA to harsh condition such as 50 mM NaOH at 65.degree. C. These
steps are not necessary using PCB-nucleotide conjugates. Second
strand synthesis and cDNA amplification is achieved by PCR in
presence of PCB-nucleotides. All the PCR products are ethanol
precipitated and are ligated with a suitably digested vector. These
vectors are rapidly purified by using magnetic capture and
illumination. These vectors can be screened using PCB-modified
hybridization probes to selectively remove the vector of interest.
This vector can then be magnetically captured and illuminated to
obtain pure vector containing the DNA of interest.
[0173] Another aspect of the invention facilitates the process of
site-directed mutagenesis. For example, cassette mutagenesis is a
powerful approach in creating site directed mutants and avoids
sequencing of entire genes to confirm the introduction of a
mutation. Basic steps in cassette mutagenesis require construction
of a vector which contains the gene of interest with
well-separated, unique restriction sites. Restriction digestion of
vector carrying the gene of interest using two unique restriction
enzymes to remove a cassette of double-stranded DNA where the
mutation is to be introduced.
[0174] The cassette containing the desired mutation is synthesized
using automated oligonucleotide synthesis. The digested vector and
the cassette are ligated to generate complete vector. These ligated
mixtures are transformed into host cells and the colonies are
screened by sequencing the cassette regions of plasmid mini-preps.
Although this process is capable of rapidly and accurately
generating a large number of site directed mutants as shown in case
of bacteriorhodopsin (H. G. Khorana, J. Biol. Chem. 263:7439,
1988), there are several areas where time and resources can be
saved using PCB.
[0175] After the initial vector restriction, the new mutant
containing cassette is labeled either chemically or enzymatically
with PCB. Subsequent ligation mixture is purified using
streptavidin interaction, for example, magnetic capture using
Dynabead-280 streptavidin. The capture results in selective
isolation of recombinant vector containing the PCB-cassette and
free PCB-labeled cassette. Photolysis releases the mutant
containing vector in pure form in any desired solution and
concentration. Subsequent transformants have very high probability
of containing only the desired mutant.
[0176] In current protocols, after restriction digestion of vector,
complete purification of doubly restricted vector by, for example,
agarose gel electrophoresis, is often difficult. The size
difference of the resulting DNA fragments is typically very small.
For example, the size of a typical cassette is about 30 base-pairs
(bp) and a typical vector about 5000 bp. Restriction enzyme
digestion to remove the cassette would produce fragments of 5000 bp
and 30 bp which can be readily distinguished and isolated. However,
this is assuming that complete digestion has occurred. Partial
digestion would produce an additional fragment of 5030 bp which is
not easily detected much less distinguished or isolated. Thus,
ineffective purification of restricted vector creates a higher
chance of ligation without incorporation of the mutant cassette.
Use of PCB avoids gel electrophoresis and subsequent elution of
restricted fragments. Magnetic capture can be performed even in the
ligation mixture.
[0177] Liposomes are widely used for targeting and introduction of
biologically important materials into cells via fusion into the
cell membrane (G. Gregoriadis editor, Liposome Technology, vol. II,
CRC Press, Boca Raton, Fla., 1984). The avidin-biotin system is
useful for in vitro studies to mediate between encapsulated
liposomes and target cells. These studies involve the use of biotin
containing phospholipids to introduce biotin into membranes (E. A.
Bayer et al., Biochim. Biophys. Acta 550:464, 1979). Avidin-biotin
system has also been attempted for targeting drugs into cells.
These studies have determined that the effects of biotinylation on
the properties of the modified lipid and biotinylated molecule,
albeit chemically altered, maintains their fundamental properties.
For example, the biotinylated lipid is fully extractable in organic
solvents, forms liposomes and mixed liposomes, and is correctly
oriented in the latter (B. Rivnay et al., Methods Enzymol. 149:119,
1987). Fatty acid components comprise the interior of the bilayer
and the biotinyl head groups are exposed to the aqueous environment
of the solvent. Thus, the use of biotinylated lipids for liposome
preparation allows an almost irreversible binding of
biotin-streptavidin interaction. Although biotin and is interaction
allows easy manipulation of these liposomes, any attempt to
concentrate or separate these liposomes using immobilized
streptavidin, such as magnetic beads coated with streptavidin,
fails as it is virtually impossible to separate the
concentrated/separated liposomes from bound streptavidin.
[0178] Use of PCB-lipids readily overcomes these limitations as
illumination releases the PCB-lipid containing liposomes into
desired solutions at desired concentrations. The steps involved in
the manipulations of liposomes using PCB-lipids include (1)
attachment of a photocleavable biotin derivative to a liposome by
chemical means or, alternatively, incorporation of a photocleavable
biotin lipid (PCB-lipid) into a liposome by first derivatizing the
lipid with PCB followed by preparation of liposome (PCB-liposome),
(2) concentration or separation of the PCB-liposome through the
selective interaction of the photocleavable biotin with avidin,
streptavidin or their derivatives which is normally immobilized on
a material such as magnetic beads, affinity column packing
materials and filters, and (3) detachment of the photocleavable
biotin from the PCB-liposome by illumination at a wavelength which
causes the photocleavable biotin covalent linkage to be broken.
[0179] The methods for attachment of various photocleavable biotins
directly to liposomes involves modification of the functional
groups on the lipids molecules using PCB. The choice of a
particular photocleavable biotin depends on which molecular groups
are to be derivatized on the lipids constituting the liposome. For
example, attachment of photocleavable biotin to a liposome could be
accomplished by forming a covalent bond with the amino group on the
phosphatidylserine. Although a number of group-specific PCB
derivatives are available that allow modification of any functional
group to yield PCB-lipid, phosphatidylethanolamine and
phosphatidylserine are exemplary. PCB-phosphatidylserine and
PCB-phosphatidylethanolamine are shown in FIG. 16.
[0180] Concentration and separation of liposome from a heterologous
mixture is readily achieved using photocleavable biotin by
established procedures that are similar to the more conventional
methods utilizing non-cleavable biotin lipids. This normally
involves an affinity technique based on streptavidin-biotin
interaction whereby the liposomes containing biotin are immobilized
due to their interaction with streptavidin. These techniques
include streptavidin-coated magnetic beads, streptavidin-sepharose
columns and streptavidin conjugated filters, all of which are
commercially available. After concentrating PCB-liposomes using
affinity interaction with streptavidin, liposomes can be released
by illumination in desired solution and at the desired
concentration.
[0181] Avidin-biotin technology is used extensively in the field of
affinity cytochemistry where specific cell structures or
subcellular components are localized by selective labeling. Two
different approaches termed immunohistochemistry (IHC) and in situ
hybridization (ISH) are available. In IHC, a primary antibody or
binding ligand which is biotinylated (alternatively, a biotinylated
secondary antibody can be utilized), is directed at a specific
antigen on the surface of a cell or cellular structure. The cell or
cellular structure is then localized by application of a reporter
complex which could consist of an avidin-enzyme conjugate,
avidin-fluorescence marker or avidin-ferritin complex for electron
microscopic localization.
[0182] In ISH, a DNA probe which is biotinylated is used to label
the location of specific mRNA or DNA sequence in individual cells
or tissue sections. A similar range of avidin based reporter
complexes can be used as in immunohistochemistry including enzyme,
fluorescence and ferritin conjugated avidins. However, a serious
limitation of the conventional application of these two techniques
is the difficulty of removing the biotin-marker complex once a
label has been applied to sample. Removal of the label would enable
additional labels to be applied thereby providing a means to map
the interrelationship between various cellular and subcellular
components in a tissue. The use of PCB provides a convenient means
to achieve this goal since photocleavage of the linker connecting
the antibody or DNA probe and PCB will result in release of the
marker complex.
[0183] In situ hybridization techniques are used to detect specific
cellular. DNA or which are non-uniformly distributed in individual
cells or tissue sections, and to detect viral nucleic acid
sequences which are often focal in distribution. In contrast to
Southern-, Northern- or dot-blot hybridization assays which
determine the average content of target molecules per cell in the
extracted tissue, ISH detects specific target sequences that are
focally distributed in a small number of cells that contain
significant levels of target molecules (E. J. Gowans et al.,
Nucleic Acid Probes, R. H. Symons editor, CRC Press, Boca Raton,
Fla., 1989).
[0184] Current technology is limited by fact that a single tissue
section, chromosome slide or cell-slide is usable only once and
probing the distribution of second target which could be another
set of sequences that are co-regulated or correlated, is almost
impossible. PCB labeled probes offer completely non-invasive
approach for multiple in situ hybridizations on such valuable
sample. Combined with multiple in situ hybridizations, a composite
picture can be constructed of distribution of various nucleic
acids. The protocol for ISH is adapted from published procedures
(D. J. Brigati et al., Virol. 126:32, 1983; I. Guerin-Reverchon et
al., J. Immunol. Meth. 123:167, 1989). The procedure involves
careful fixation and sectioning of tissues which may be either
paraffin or frozen sections. Fixation is designed not only to fix
nucleic acids, but also to bind the section firmly to the slide.
Glutaraldehyde is used for DNA and paraformaldehyde is used for RNA
detection. Both steps include optional denaturation steps which are
necessary if dsDNA or dsRNA is the target of reaction. Further
steps involve preparation of different probes that are labeled
using PCB, and hybridization of these probes in a sequential
manner. ISH follows the same general principles as a solution and
filter hybridization (R. J. Britton et al., Nucleic Acid
Hybridization, B. D. Hames and S. J. Higgins editors, IRL Press,
Oxford, 1985). PCB-labeled probe is then detected using variety
techniques that use streptavidin conjugated detecting systems. A
picture is obtained that shows distribution of first probe in the
tissue section or chromosome picture. Photolysis results in release
of detection assembly along with biotin moiety. ISH and detection
is then repeated with second probe and its distribution is
obtained. A composite picture is created that shows distribution of
a variety of probes in the tissue section.
[0185] In situ hybridization (ISH) techniques are also used to
detect either specific cellular DNA or RNA sequences at the
chromosomal level in individual cells or tissue sections. The
method is referred to as hybridization histochemistry. ISH is
ideally suited not only to the detection of cellular nucleic acid
sequences which are non-uniformly distributed in tissues or cell,
but also to the detection of viral nucleic acid sequences which are
often focal in distribution. The detection of nucleic acid by ISH
satisfies the primary objective of reflecting accurately the
intercellular and intracellular distribution of target molecules in
the sample. In general, ISH may be used to detect DNA corresponding
to normal or abnormal genes, to identify the chromosomal location
of particular DNA sequences, and to measure the level of expression
of these genes by mRNA detection.
[0186] In particular, mRNA is a common target for in situ
hybridization reaction in studies of gene expression and cell
differentiation. In the special cases of virus infected cells,
either viral genomic nucleic acid or RNA transcripts can be
detected. ISH is especially valuable where the histological mapping
of target cells within a tissue is sought. In contrast, Southern,
Northern and dot-blot hybridization assays measure the overall
concentration of the target molecules per cell in the extracted
tissue. If PCB is substituted for biotin in the application of
immunochemistry and in situ hybridization, the methodology for
localization of a label is almost identical. However, an important
advantage is the ability to completely remove the label in the form
of a PCB-avidin marker complex by simple illumination of the sample
which photocleaves the PCB linkage to the antibody or hybridization
probe. This step renders the sample, typically a tissue section,
available for further sequential labeling by additional different
probes.
[0187] There is often a need to determine if antigenic peptides,
hormones or viral gene products detected intracellularly by
immunohistochemical techniques represent de novo synthesis or
merely deposition and passive accumulation. It is also useful to
determine if specific mRNA, detected by ISH is translated into
protein product. The use of PCB facilitates such a determination
since sequential interrogation of a single sample is possible using
the same enzyme-avidin linked reporter complex. This approach
avoids complications due to the use of different samples.
[0188] Many different genes, difficult or impossible to locate
otherwise, can be localized using conjugates of the invention.
Neurotransmitter receptors are members of large gene families. By a
combination of expression strategies and homology cloning, dozens
of receptor genes in this family have now been cloned. These genes
include those encoding receptors for glutamate, glycine, and
.gamma.-aminobutyric acid (GABA) receptors. Cloning has revealed
the existence of distinct neurotransmitter receptors in numbers
that had not been anticipated by neurophysiologists. The
significance of this surprising receptor homogeneity is not yet
known but ISH has shown that individual receptor subtypes are
expressed in unique patterns in the brain. An important goal is to
determine the distribution of these receptors in the brain.
Ordinarily, this is done using many thin slices each exposed to a
different hybridization probe. However, these experiments suffer
from the use of multiple tissue sections. In contrast, the use of
PCB would allow repeated analysis of the same rat brain section to
determine complete distribution of each receptor expression.
[0189] Further, cloned genes and markers can be localized by ISH to
specific regions of chromosomes. Sequences can be localized to
sub-chromosomal regions by hybridizing radioactively labeled probes
directly to chromosome spreads. Chromosome spreads are made by
using cells whose division has been blocked in the metaphase by a
chemical like colcemid which disrupts the mitotic spindle. After
fixing and staining, a pattern of light and dark bands develops on
each chromosome, so that the chromosomes can be identified. After
ISH, location of the radioactive probe is revealed by the
distribution of silver grains in a photographic emulsion layered
over the spread. However, the detection of single copies of human
genes is difficult and can be done only by pooling the distribution
of grains over as many as 30 or more metaphase spreads.
[0190] PCB offers the unique advantage that the probe can be
photoreleased after localization of a specific genetic sequence on
a chromophore and a second probe applied. In cases where too
closely spaced sequences are to be detected, prior removal of the
probe may be essential in order to avoid interference from the
original probe. In addition, the method allows for the use of a
large number of probes to be applied sequentially and is not
limited by the availability of different marker complexes which can
be simultaneously measured. This technique could also be used
advantageously for rapid ordering of multiple probes on a
chromosome.
[0191] An important application of biotin-avidin technology is the
separation of cells from a complex mixture often containing a
variety of different cell types. Cells which can be utilized
include cells within a biological sample, tissue culture cells,
bacterial cells and diseased cells. Cells may be mammalian, such as
mammalian stem or fetal cells. Receptors include antibodies
directed against the classical cell surface receptors and other
surface proteins, but also any cell-surface molecule that has a
specific affinity for another molecule. Preferably, the receptor is
an antibody which recognizes a cell surface marker on the target
cell or a specific protein which recognizes a cell surface ligand
or other macromolecule on the target cell. PCB-modified antibodies,
specific for surface antigens, receptors or ligands on the cell,
can be used for cell separation or cell sorting with, for example,
an apparatus such as a fluorescence-activated cell sorter (FACS).
These PCB-antibodies, when linked to streptavidin-coated magnetic
beads can bind to the specific cell population bearing a particular
antigen and can then be separated using Immunomagnetic Separation
(IMS).
[0192] Current methodologies do not allow gentle separation of
these magnetic particles from separated or sorted cell population.
PCB modified antibodies, however, can be readily separated from
magnetic particle after illumination IMS involving PCB-antibodies
can be used in cell-sorting, tissue typing and for selective
enrichment of microorganisms using modifications of protocols
described earlier (A. Elbe, J. Immunol. 149:1694, 1992; S. Qin,
Sci. 259:974, 1993; L. Leclerecq, Immunol. Lett. 28:135, 1991).
[0193] In conventional methods, a biotinylated antibody is utilized
which binds selectively to an antigen residing only on the target
cell. The target cells can then be isolated from the mixture by
using streptavidin-coated magnetic beads or streptavidin based
affinity columns. The affinity material sometimes contains a
secondary antibody directed toward the primary antibody. A severe
limitation of this approach is the difficulty of releasing the
cells once they are bound to the affinity medium through the
biotin-streptavidin interaction. Normal methods that are designed
to disrupt the biotin-streptavidin interaction or the
antibody-antigen interaction can reduce the overall viability of
the released cells. This is a serious disadvantage if the cells are
to be used later for culturing or transplantation. Similarly,
conditions such as low pH that disrupt the antibody-antigen
interaction can cause cell damage. A standard technique is
overnight incubation in a culture medium followed by vigorous
mixing. This causes shedding of the antigen involved in binding.
However, this method is time consuming, can lead to cell
degradation and does not result in complete release. An alternate
method is to disrupt the antibody-antigen interaction with
enzymatic treatment, which can also be damaging to a cell. An
additional method is the utilization of anti-FAB antibodies to
compete for the antigen. Such methods are time consuming, expensive
due to the use of antibodies and only partially effective. These
methods of detachment (release) of cells are all particularly
ineffective when the antibody-antigen interaction is strong or the
binding involves several antigen-antibody interactions mixing.
Conventional biotins which can be chemically cleaved such as
NHS--SS-Biotin (sulfosuccinimidyl 2-(biotinamido)
ethyl-1,3-dithiopropionate; Pierce Chemical; Rockford, Ill.) pose
serious problems since the cleavage medium consisting of a high
concentration of reducing agents such as thiols will cause
reduction of protein disulfide bonds and a subsequent loss of cell
viability.
[0194] In contrast to conventional methods, photocleavable biotin
offers an inexpensive, effective and rapid means to release
immobilized cells simply by using light exposure. Release occurs
due to photocleavage of the covalent linkage between the antibody
and biotin. While the antibody still remains bound on the released
cell this is normally not a problem for cell integrity or
additional utilization of the separated cells. Since photocleavage
can be performed in short periods and results in almost full
removal of the photocleavable biotin release is rapid and
complete.
[0195] Further, using photocleavable conjugates, cells which
represent a very small populations of sample of cells can be
accurately and efficiently selected. This enables methods such as
the selection of immune cells, stem cells, fetal cells, precursor
cells and nearly and cell type from lymph (e.g. interstitial
lymphatic), blood (e.g. arterial and peripheral blood) or tissue
(organ, soft tissues, muscle) samples. Selected and isolated cells
can then be cultured in very large numbers and possible
reintroduced into the same or another patient. Cultured cells can
also be used in gene therapy by the introduction of genetic
material into cultured cells. Such techniques are not possible
using conventional detection and isolation procedures.
[0196] The same approach can also be used by linking photocleavable
biotin to other cell specific macromolecules such as
cell-associated ligands or antigens. For example, B cells
expressing a specific immunoglobulin receptor for a target antigen
could be isolated by attaching photocleavable biotin to the target
antigen and then binding it to a streptavidin-coated bead.
Alternatively hybridoma screening and stem cell selection could be
performed using this approach.
[0197] The three basic steps involved in cell isolation using PCB
are (1) attachment of a photocleavable biotin, or molecule
containing a photocleavable biotin derivative including
(antibodies, receptor ligands, antigen) to the surface of the
target cell by a photochemically cleavable bond, (2) separation of
said cell type from other cells and materials in the complex
mixture through the selective interaction of the photocleavable
biotin with avidin, streptavidin or their derivatives, and (3)
detachment of the photocleavable biotin from the said cell type by
illumination at a wavelength which causes the photocleavable biotin
covalent linkage to be broken. These steps, using PCB, can be
carried out in combination with ordinary biotin. In this case,
cells containing two or more surface markers can be from those
containing only one. Each of these steps is illustrated in FIG.
17.
[0198] Several applications for the use of PCB for cell separation
offers clear advantages over existing technology. A common method
for isolation of cells is to label the cell with a fluorescent dye
which is directed to the target cell through an
antibody-fluorescent conjugate. The cells can than be separated by
using a fluorescence-activated cell sorting device and used for a
variety of purposes including cell typing, hybridoma production and
tissue culturing. However, with this technique the separated cells
remain labeled with the fluorescent dye. This can result in
reduction in cell viability, prevent their use in therapeutic
applications where the dye can be toxic, and prevent further
fluorescence based sorting into subspecies of cells. One example
would be the separation of lymphocytes using characteristic
antigens such as CD2, CD4, CD8 and CD19 followed by sorting into
subspecies.
[0199] An alternative approach is to utilize an antibody which is
conjugated to PCB. In this case the cell can be fluorescence
labeled with an avidin-fluorescein complex which is commercially
available in a variety of forms (Table 8). The fluorescent label
can then be removed by photocleavage of the PCB which results in
release of the PCB-avidin-fluorescent complex. A variety of
fluorescent labels exist which absorb in a region outside of the
absorption band of the PCB, thus avoiding photocleavage during the
cell sorting.
TABLE-US-00008 TABLE 8 Commercially Available Avidin-Florescent Dye
Complexes Conjugate Absorption (nm) Emission Producer Avidin- 490
520 Pierce Fluorescein Avidin-R- 450-470 574 Pierce Phycoerythrin
Avidin- 515-520 575 Pierce Rhodamine Avidin- 575 600 Pierce
Rhodamine 600 Avidin-Texas 595 615 Pierce Red
[0200] Another example of the utility of PCB methodology for cell
isolation is the isolation of B cells for hybridoma production.
Briefly, formation of hybridomas for the purpose of monoclonal
antibody production involves the fusion of myeloma cells with
lymphocyte B cells. Generally, a heterogeneous population of B
cells is utilized which contains different immunoglobulin
receptors. The hybridoma which expresses the desired antibody is
then screened by assaying for binding to a particular antigen. This
screening process can be time-consuming and expensive. Screening
could be avoided if a method existed for isolation of a particular
population of B cells which only expressed the desired
immunoglobulin receptor. In principle, this could be accomplished
by using a biotinylated antigen, such as a polypeptide with a
specific sequence, which will only bind to those B cells which
express the specific immunoglobulin receptors for the antigen. This
subpopulation of B cells could then be selected by using avidin
affinity techniques such avidin-coated magnetic beads. However, the
subsequent step of hybridoma formation will still be prevented
unless the B cells can be released from the avidin-biotin complex
in a viable form.
[0201] The use of PCB-biotin avoids this problem by providing a
simple method for releasing the B cells in a viable form after
immobilization by the biotin-avidin interaction. Photocleavage of
the photocleavable biotin linkage with the antibody results in the
release of the B-cells and the bound antigen which is now in an
unmodified form. Since no chemical treatment is required, the cells
will retain their viability for further fusion to the myeloma
cells. Furthermore, the method is rapid and suitable for
automation.
[0202] Another embodiment of the invention is directed to targets
isolated by the above method which may be utilized in
pharmaceutical compositions to treat or prevent diseases and
disorders. Pharmaceutical compositions may comprise the isolated
targets plus a pharmaceutically acceptable carrier such as water,
oils, lipids, saccharides, polysaccharides, glycerols, collagens or
combinations of these components. The composition is administered
to patients for the treatment or prevention of certain diseases and
disorders and for the site-directed administration of
pharmaceutical agents.
[0203] Another embodiment of the invention is directed to a method
for determining an in vivo half-life of a pharmaceutical in a
patient. Conjugates are formed by coupling the pharmaceutical to a
bioreactive agent via a covalent bond that can be selectively
cleaved with electromagnetic radiation. Conjugates are administered
to the patient after which, two or more biological samples are
removed. Samples are treated with electromagnetic radiation to
release the pharmaceutical from the bioreactive agent, the amount
of the bioreactive agent in the biological samples is determined,
and the in vivo half-life of the pharmaceutical determined.
[0204] The pharmaceutical may be a composition comprising
cytokines, immune system modulators, agents of the hematopoietic
system, chemotherapeutic agents, radio-isotopes, antigens,
anti-neoplastic agents, recombinant proteins, enzymes, PCR
products, nucleic acids, hormones, vaccines, haptens, toxins,
antibiotics, nascent proteins, synthetic and recombinant
pharmaceuticals, and derivatives and combinations of these
components. Conjugates may be administered to patients by
parenteral administration, sublingual administration, enteral
administration, pulmonary absorption, topical application and
combinations thereof. Animals which can be tested include mammals
such as humans, cattle, pigs, sheep, dogs, cats, horses and
rodents. Biological samples which are collected can be sample of
peripheral blood, blood plasma, serum, cerebrospinal fluid, lymph,
urine, stool, ophthalmic fluids, organs and bodily tissues.
[0205] Another embodiment of the invention is directed to the
controlled release of a substrate into a medium. Conjugates
comprised of a bioreactive agent coupled to the substrate by a
covalent bond which can be selectively cleaved with electromagnetic
radiation are created as described. These conjugates are bound to a
surface of an article and placed into the medium. The surface of
the article is exposed to a measured amount of electromagnetic
radiation and the substrate released into the medium to carry out a
beneficial effect. Alternatively, an article may be placed at a
selected site and the conjugates, having an affinity for the
article, are administered at a distal site. Conjugates then migrate
to the selected site and perform an intended function. After
completion of that function, radiation is applied and the substrate
is released from the fixed bioreactive agents. Released substrate
may be naturally eliminated from the patient's system. This can be
highly useful, for example, in radiation therapy for cancer
patients.
[0206] Preferred are radiation wavelengths which can penetrate the
medium. Depending on the amount and frequency of radiation
exposure, release can be controlled and continued over a period of
time. This method is useful for the controlled and site-directed
administration of pharmaceutical compositions to a patient. In such
cases, the medium in which the article is placed may be blood,
lymph, interstitial fluid or a tissue. Controlled release may also
be performed in tissue culture for administering a constant or
periodic amount of a substrate to a cell culture fluid or balanced
salt solution for uptake by the cells. Articles which may be
coupled with substrate and placed within or adjacent to a patient's
body include articles comprising carbohydrates, lipids, proteins,
polysaccharides, cellulose, metals including magnets, organic
polymers and combinations thereof. Preferably, the surface of the
article is coated with streptavidin and the bioreactive agent is
photocleavable biotin.
[0207] Alternatively, articles containing conjugates or agents can
be placed into the site of the disorder, such as a tumor. The
pharmaceutical agent such as, for example, a radioactive agent is
administered to the patient and becomes bound to the fixed
conjugates or agents. Effects attributable to the pharmaceutical
agent are localized. The article is exposed to a measured amount of
electromagnetic radiation and the pharmaceutical agents released
into the body and excreted. This method is preferred when only a
short term exposure of the pharmaceutical agent is desired or to
efficiently remove potentially harmful agents after they have had
their desired effects.
[0208] Another embodiment of the invention is directed to a method
for creating a photocleavable oligonucleotide. A bioreactive agent
is created comprised of a photoreactive moiety coupled to a
detectable moiety and containing a phosphoramidite. The
oligonucleotide is synthesized using conventional phosphoramidite
chemistry. The nucleotide precursors comprise one or more
photocleavable phosphoramidites such as purine-phosphoramidites
(uracil, cytosine, thymine) or pyrimidine-phosphoramidites
(adenine, guanine) as the ribose or deoxyribose forms, or
derivatives thereof. This method can be performed manually or
automated using a commercially available oligonucleotide
synthesizer. Photocleavable oligonucleotides can be utilized as
primers or probes, in diagnostic kits and in every instance in
which a nucleic acid can be used.
[0209] Another embodiment of the invention is directed to a method
for detecting a target molecule in a heterologous mixture.
Conjugates are formed by coupling a substrate to a bioreactive
agent with a covalent bond that is selectively cleavable with
electromagnetic radiation. Conjugates are contacted with the
heterologous mixture to couple substrate to one or more target
molecules. Uncoupled conjugates are removed and the coupled
conjugate are treated with electromagnetic radiation to release the
detectable moiety. Presence of target molecules can be detected by
detecting the presence of the released detectable moiety. Target
macromolecules may be proteins, peptides, nucleic acids, lipids,
polysaccharides, metallic compounds, virus, bacteria, eukaryotic
cells, parasites and derivatives and combinations thereof. Once
detected, target macromolecules can be isolated and the amount
isolated quantitated by current any of the techniques available to
those of ordinary skill in the art.
[0210] A specific application of streptavidin-biotin technology is
in the detection of targets in medical diagnosis. Generally, the
target is a biotinylated molecule or a biotinylated probe for the
target molecule. The interaction of streptavidin with the
biotinylated target or probe is amplified by an enzyme conjugated
to streptavidin which catalyzes a chromogenic reaction. For
example, a variety of enzymes conjugated to streptavidin are
commercially available including horseradish peroxidase,
.beta.-galactosidase, glucose oxidase and alkaine phosphate. Each
of these enzymes catalyze a chromogenic reaction. Additional
methods of detection include conjugating streptavidin to
fluorescent, chemi-fluorescent, radioactive or electron dense
molecules.
[0211] An example of biotin-avidin interaction in medical diagnosis
also forms the basis for a wide array of enzyme-linked
immunospecific assays (ELISA). In this case, an antibody specific
for the target molecule, the primary antibody, or a secondary
antibody directed against the primary antibody is biotinylated.
Detection is accomplished with a streptavidin-enzyme conjugate as
described. A large number of immunoassays have been developed based
on this approach. However, multiple immunoassays on the same sample
are not easily accomplished using conventional technology since
there exists no simple method of removing the avidin-enzyme-complex
once bound to a biotin derivative without damaging the sample. In
contrast to traditional methodologies, PCB can be reused many times
for multiple assays of the same sample for multiple screening of
pathogens and other markers of human diseases.
[0212] The biotin-avidin interaction also forms the basis of
sensitive methods for detecting specific nucleic acid sequences
such as screening human DNA samples. DNA or RNA probes which
hybridize to a target DNA sequence are biotinylated. DNA containing
the target sequence is detected with an streptavidin-enzyme
conjugate. This method is used in conjunction with the polymerase
chain reaction where the target gene or sequence to be screened is
first amplified using specific primers. The introduction of biotin
nucleotides into the primer or directly into the nascent PCR
product using biotinylated nucleotides facilities isolation of the
target DNA. A variety of biotinylated nucleotides such as
biotin-dUTP are available for such purposes. However, this method
has at least two limitations.
[0213] First, the presence of biotin in the amplified target DNA
prevents the use of biotinylated probes without prior removal of
the biotinylation. The presence of endogenous biotin or
biotin-containing molecules in the sample also lowers the
sensitivity of this assay. Second, the presence of biotin in the
target DNA lowers hybridization efficiency and hence the
sensitivity of the assay. As discussed above, the use of PCB
derivatives such as PCB-nucleotides effectively eliminates these
problems by allowing for complete removal of biotin from the
procedure.
[0214] Conjugates and methods of the invention can be used in
conjunction with a variety of diagnostic assay involving nascent
protein detection. For example, diagnostic assays for cancer have
been developed which rely on in vitro expression of PCR amplified
genes followed by examination of the nascent protein product using
gel electrophoresis (S. M. Powell et al., N. Engl. J. Med.
329:1982, 1993). The isolation of such proteins and the subsequent
sensitivity of such tests could be increased by the incorporation
of PCB amino acids.
[0215] Biotin-streptavidin technology is widely used as the basis
for non-radioactive ELISA including diagnostic assays for specific
indicators of diseases and disorders such as disease-linked
antigens including adenovirus antigen (K. Mortensson-Egnund et al.,
J. Virol. Methods 14:57, 1986), bovine leukemia virus (E. N.
Esteban et al., Cancer Res. 45:3231, 1985), flavivirus (E. A. Gould
et al., J. Virol. Methods 11:41, 1985), Hepatitis B surface antigen
(C. Kendall et al., J. Immunol. Methods 56:329, 1983), Herpes
simplex virus antigen (K. Adler-Strorthz et al., J. Clin.
Microbiol. 18:1329, 1983), respiratory syncytial virus (A.
Hornsleth et al., J. Med. Virol. 18:113, 1986), bacterial antigens
(R. H. Yolken et al., J. Immunol. Methods 56:319, 1983) and
melanoma-associated antigens (human) (A. C. Morgan et al., Cancer
Res. 43:3155, 1983). The usefulness of these assays can be
compromised if endogenous biotin is present in the sample. In this
case, a false background will be obtained since the
streptavidin-reporter enzyme complex will react both to
non-specific biotins and to the biotinylated antibodies directed
against the target protein. While several approaches to eliminate
background due to non-specific binding of the avidin or
streptavidin to non-biotinylated targets including the use of high
ionic-strength buffers (C. J. P. Jones et al., Histochem. J.
19:264, 1987), milk proteins (R. C. Duhamel et al., J. Histochem.
Cytochem. 33:711, 1985) and lysozyme (E. A. Bayer et al., Anal.
Biochem. 163:204, 1987) and altered streptavidins such as
ImmunoPure NeutrAvidin (Pierce Chemical; Rockford, Ill.), none has
been very effective in eliminating background due to endogenous
biotin.
[0216] Use of photocleavable biotins in conjunction with
immunoassays alleviates the problem by providing a means of
determining the background level of non-specific biotin. The ELISA
assay is first performed using conventional methodology except that
the antibody directed against the protein is targeted with
photocleavable biotin (FIG. 19). The streptavidin-reporter enzyme
complex linked to the probe antibody system is then removed via
light cleavage of the photocleavable biotin Cleavage does not
release the reporter enzyme-avidin complex bound from non-specific
bound biotin since this biotin is non-cleavable. The signal
obtained from the remaining biotin can be used as a measure of the
endogenous biotin present in the sample and subtracted from the
primary signal obtained in step 1.
[0217] Background signal due to endogenous biotin in a
biotin-avidin based ELISA can be simply detected using
photocleavable biotin The ELISA is performed according to normal
procedures using streptavidin-reporter enzyme complex. However, the
streptavidin-reporter enzyme complex is then removed with light and
the system reassayed to determine the background level of
endogenous biotin.
[0218] A second application of photocleavable biotins is its use to
conduct multiple biotin based ELISA assays on the same sample. This
is based on the ability to fully remove the streptavidin-reporter
enzyme complex with light upon photocleavage of the PCB linkage. In
this case a second ELISA can be performed using a different
antibody probe and reporter enzyme complex without interference
from the first ELISA as shown in FIG. 20. Normally such a multiple
ELISA is not possible because the signal obtained from a second
antibody probe combined with a streptavidin-reporter enzyme complex
cannot be easily separated from the original signal.
[0219] For example, in a conventional biotin-avidin ELISA, the
streptavidin-reporter enzyme complex remains bound to the probe
antibody even after the chromogenic product of the reporter enzyme
is removed. Thus, it will continue to produce a chromogenic product
even after a second ELISA is performed and interfere with the
signal of that second immunoassay. In contrast, the removal of the
streptavidin-enzyme complex by cleavage of the photocleavable
biotin with light and then subsequent washes eliminates this
problem since the original reporter enzyme is no longer
present.
[0220] In another application of the invention, detection of
pathogens such as microorganisms from biological material often
requires their isolation and culturing. The more effective the
isolation step, the more reliable and rapid the culturing step will
be because of the elimination of other contaminants and the
concentration of the target pathogen. While a variety of affinity
techniques exists for separation of microorganisms such as magnetic
beads conjugated to selective antibodies, the problem of release of
the microorganisms in a viable form suitable for culturing and
sensitive detection still remains. In contrast, PCB which is linked
to the antibody or binding ligand provides a non-damaging and rapid
means for photochemical release of the microorganism in a viable
form.
[0221] For example, this application of the invention provides the
basis for development of rapid diagnostic assays for a variety of
pathogens involved in human and animal disease that were previously
not possible using conventional biotin-streptavidin technology.
Microorganisms could also be isolated from food, milk, soil and
other materials for the purpose of depletion or detection using
this approach.
[0222] Another embodiment of the invention is directed to methods
for treating a disorder by the controlled release of a therapeutic
agent at a selected site. Conjugates are formed by binding a
bioreactive agent to a therapeutic agent with a bond that is
selectively cleavable with electromagnetic radiation, wherein the
bioreactive agent is comprised of a directable moiety bonded to a
photoreactive moiety and the directable moiety has an affinity for
the selected site. Conjugates are administered to a patient having
the disorder and the selected site is subjected to a measured
amount of electromagnetic radiation for the controlled release of
the therapeutic agent to treat the disorder. Disorders which can be
detected include infections, such as bacterial infections, viral
infections and parasitic infections, neoplasias such as a tumor,
and genetic disorders such as an overproduction or deficiency of an
enzyme or other genetic product.
[0223] The therapeutic agents may be toxins, immune system
modulators, hematopoietic agents, proteins, nucleic acids,
substrate analogs, transcription and translation factors, antigens
and combinations thereof. Directable moieties may be antibodies
such as a monoclonal or polyclonal antibody or antibody
fragment.
[0224] Another embodiment of the invention is directed to
diagnostic kits for detecting or screening for diseases and
disorders in patients. Kits contain a conjugate comprised of a
bioreactive agent covalently bonded to a diagnostic agent having an
affinity for an indicator of said disorder in a biological sample
obtained from the patient. The indicator may be a presence or
absence or an increased or decreased amount or level of a
characteristic marker of the disorder such as an antigen or
antibody, a cytokine, a specific cell type (e.g. B cells;
cytotoxic, suppressor or helper T cells; macrophages; stem cells),
a particular enzyme, nucleic acid or protein. Disorders which can
be detected include infections, neoplasias and genetic disorders.
Infections which can be detected include bacterial infections,
viral infections and parasitic infections. Neoplasias which can be
detected include tumors. Genetic disorders which can be detected
include an overproduction or deficiency of an enzyme. Biological
samples which can be added to the sample include samples of
peripheral blood, blood plasma, serum, cerebrospinal fluid, lymph,
urine, stool ophthalmic fluids, organs and bodily tissues. Such
kits may also be used to detect or screen for the presence of fetal
or stem cells in a biological sample which can be isolated and
cultured or further analyzed.
[0225] Kits may also be used to detect the presence of multiple
nucleic acids and/or proteins on, for example, an electroblot using
a series of secondary probes linked to biotin. After each probe is
introduced, the biotin attachment could be cleaved allowing the
enzymatic assay complex to be removed thus providing for a new
secondary probe to be introduced. Such an approach would be
extremely useful as the basis of medical diagnostic assays, where
multiple antigens or nucleic acid sequences needed to be probed
rapidly and automatically.
[0226] The kit may also be a nucleic acid mutagenesis kit for use
in molecular biological applications such as introducing or
correcting mutations in DNA or RNA.
[0227] The nucleic acid may be an oligonucleotide for use in PCR or
cassette-type applications. Such oligonucleotides may be
single-stranded or double-stranded and preferably contain one or
more restriction enzyme recognition sequences internally and
ligatable 5' and 3' ends which also contain part of a restriction
enzyme recognition site. Alternatively, one or more ends may be
blocked to facilitate directed coupling.
[0228] The following examples are offered to illustrate various
embodiments of the invention, but should not be viewed as limiting
the scope of the invention.
EXAMPLES
Example 1 Synthesis of Photocleavable Agents
[0229] Five grams or 27.6 mmol of 5-methyl-2-nitrobenzoic acid
(FIG. 5, compound "6"; Aldrich Chemical; Milwaukee, Wis.) was added
in small portions to 10 ml (16.4 g or 148 mmol) of thionyl chloride
with stirring. The mixture was stirred at room temperature for 10
hours. Excess of thionyl chloride was removed by vacuum to give the
acid chloride ("7"). Magnesium turnings (1.07 g or 44.2 mmol),
absolute ethanol (6 ml), chlorobenzene (8 ml), and 0.1 ml of dry
CCl.sub.4, were refluxed until most of the magnesium reacted. A
solution of diethyl malonate (4.82 g or mmol) in 10 ml of
chlorobenzene was added followed by the addition of the acid
chloride (5.49 g) in 10 ml of chlorobenzene. The reaction mixture
stirred for 1 hour and 1.7 ml of concentrated H.sub.2SO.sub.4 in 17
ml of H.sub.2O was added, stirred for additional 15 minutes. 20 ml
of chloroform was added and the layers separated. The aqueous layer
was extracted three times with 10 ml and the extracts were
combined, dried and evaporated to dryness. Residue was dissolved in
8.25 mls of acetic acid. 5.4 ml of H.sub.2O and 1 ml of
concentrated H.sub.2SO.sub.4 were added, the mixture was refluxed
for 6 hours, neutralized with aqueous Na.sub.2CO.sub.3 and
extracted three times with 20 ml of CHCl.sub.3. Extracts were
combined, dried and solvents removed by vacuum. Residue was
crystallized from 70% ethanol to produce 4.46 g, or about 81%, of
5-methyl-2-nitroacetophenone. 5-methyl-2-nitroacetophenone (3.5
.mu.g or 19.6 mmol), N-bromosuccinimide (3.66 g or 20.6 mmol), and
benzoyl peroxide (46 mg or 0.01 eq) were refluxed in 20 ml of CCl,
for 5 hours. The reaction mixture was filtered, the filtrate
concentrated and crystallized from CCl.sub.4 to produce 3.64 g
(72%) of 5-bromomethyl-2-nitroacetophenone ("8"). Compound 8 (2.0 g
or 7.75 mmol) was added to a solution of hexamethylenetetramine
(1.14 g or 8.13 mmol) in 15 ml of chlorobenzene. The mixture was
stirred overnight, the precipitate filtered off and washed with 10
mls of chlorobenzene and 20 mls of diethyl ether. The precipitate
(2.93 g or 736 mmol) was suspended in 35 ml of 95% ethanol followed
by the addition of concentrated HCl (3.12 ml or 5 eq.). The mixture
was stirred overnight and evaporated to dryness. Ten mls of DMF
were added to the residue followed by the addition of a
6-biotinamidocaproic acid (3.29 g or 1.25 eq.) in 35 ml of
N-methylpyrrolidone, dicyclohexylcarbodiimide (228 g or 1.5 eq.),
and triethylamine (1.28 ml or 1.25 eq.). The solution was stirred
overnight at room temperature, the precipitate filtered off, and
filtrate precipitated to 700 ml of diethyl ether. The precipitate
was dried and purified on a silica gel column using step gradient
(5-20%) of MeOH in CHCl.sub.3 to produce 2.27 g (about 58%) of
compound 11.
[0230] Compound 11 (1 g or 1.87 mmol) was dissolved in 15 ml of 70%
EtOH (FIG. 5). The solution was cooled to 0.degree. C. and sodium
borohydride (141 mg or 4 eq.) added. The solution was stirred at
0.degree. C. for 30 minutes and at room temperature for an
additional 2 hours. The reaction was quenched with the addition of
1 ml acetone, neutralized with 0.1N HCl, concentrated, the
supermatant discarded, the residue washed with water and dried to
produce 0.71 g (about 71%) of compound 12.
[0231] Compound 12 (1.07 g or 2 mmol) was dissolved in 10 ml DMF.
N,N'-disuccinimidyl carbonate (Fluka Chemical; Ronkonkoma, N.Y.) (1
g, 15 eq.) was added followed by 0.081 ml or 3 eq. of triethylamine
(FIG. 5). After 5 hours at room temperature, solvents were
evaporated to dryness and the residue was washed consecutively with
0.1N NaHCO.sub.3, water, dioxane, diethyl ether and dried to give
1.04 g (about 69%) of 5-(5-biotinamidocaproamidomethyl)-1-(2-nitro)
phenylethyl-N-hydroxysuccinimidyl carbonate (PCB-NHS) ester
(compound 13). M.P.=113-114.degree. C. (uncorrected); CI-MS
(M.sup.+=676.5); UV-VIS.lamda.=204 nm, .epsilon.1=19190 M.sup.-1
cm.sup.-1; .lamda.2=272 nm, .epsilon.2=6350 M.sup.-1 cm.sup.-1 in
phosphate buffer, pH=7.4. .sup.1H NMR (DMSO-d.sub.6, Varian XL-400
MHz), [.delta.ppm]: 8.48 (t, 1H), 8.05-8.03 (d, 1H), 7.75-7.71 (t,
1H), 7.66 (s, 1H), 7.46-7.45 (d, 1H), 6.44 (s, 1H), 6.37 (s, 1H),
6.28-6.27 (m, 1H), 439 (m, 2H), 4.30 (m, 1H), 4.12 (m, 1H), 3.57
(d, 2H), 3.09 (m, 1H), 3.01-2.99 (m, 2H), 2.79 (m, 5H), 258-2.55
(d, 1H), 2.17-2.15 (m, 2H), 2.04-2.02 (m, 2H), 1.72-1.71 (m, 2H),
1.66-1.43 (m, br, 6H), 1.38-1.36 (m, br, 2H), 1.26-1.25 (m, br,
3H); IR (KBr); Vc=o 1815 and 1790 cm.sup.-1.
[0232] Synthesis of PCB-NHS ester (FIG. 18):
2-Bromo-2'-nitroacetophenone ("14") (Aldrich Chemical; Milwaukee,
Wis.) (1 g; 4.09 mmol) was converted into
2-amino-2'-nitroacetophenone hydrochloride ("15") by reaction with
1.05 eq. of hexamethylenetetramine and hydrolysis, and was coupled
to 5-biotinamidocaproic acid (1.25 eq.) using DCC (1.5 eq.) in DMF
to produce 2-(5-biotinamidocaproarrnido)-2'-nitroaceptophenone
("16") (about 52% yield) which was reduced (about 75% yield;
"17"and converted into reactive NHS derivative ("18")
2-(5-biotinamidocaproamido)-2'-nitrophenylethyl-N-hydroxysuccinimidyl
carbonate (about 69% yield) as described.
[0233] Synthesis of PCB-NHS ester (FIG. 19):
3-amino-4-methoxybenzoic acid ("19") (Aldrich Chemical; Milwaukee,
Wis.) (5 g or 29.9 mmol) was suspended in 40 ml acetic acid. Acetic
anhydride (3 ml or 1:04 eq) was added by stirring. The reaction
mixture was stirred for 2 hours at room temperature. 25 ml of 0.1N
HCl was added and the precipitate was filtered off and washed with
3.times.10 ml of 0.1N HCl and 5.times.10 ml water to produce 5.97 g
(about 95%). 3-(N-acetyl) amino-4-methoxybenzoic acid ("20") (5 g
or 235 mmol) was added to 20 ml of fuming nitric acid at 0.degree.
C. on vigorous stirring. The solution was stirred at 0.degree. C.
for an additional hour and poured onto 200 g of ice. Precipitate
was filtered off, washed with 5.times.20 mls of water and dried to
produce 438 g (about 72%) of 2-nitro-4-methoxy-5
(N-acetyl)aminobenzoic acid ("21"), which was converted into
2-nitro-4-methoxy-5-(N-acetyl)aminoacetophenone ("22") (about 63%
yield) as described.
2-nitro-4-methoxy-5-(N-acetyl)aminoacetophenone ("22") (1 g or 4.76
mmol) was dissolved in 5 ml of DMF and the solution was added to
5-biotinamidocaproyl chloride prepared separately from
5-biotinamidocaproic acid (1.55 g or 1 eq.) and 5 ml thionyl
chloride. The reaction mixture was stirred overnight at room
temperature and added to 100 ml of diethyl ether. Precipitate was
purified using small silica gel column and a step gradient of MeOH
in CHCl.sub.3 to give 1.16 g (about 47%) of
2-nitro-4-methoxy-5-(5-biotinamidocaproamido) acetophenone ("23").
This intermediate was converted into its corresponding alcohol
(about 85% yield) and into the reactive NHS ester derivative
5-(5-biotinamidocaproamido)-4-methoxy-1-(2-nitro)phenylethyl-N-hydroxysuc-
cinimidyl carbonate ("25") with about a 69% yield.
[0234] Synthesis of photocleavable coumarin (FIG. 21):
5-aminomethyl-2-nitroacetophenone hydrochloride ("26") (1.15 g or 5
mmol) was dissolved in 20 ml DMF. To this solution
7-Methoxycoumarin-4-acetic acid ("27") (Aldrich Chemical;
Milwaukee, Wis.) (1.46 g or 1.25 eq.) and dicyclohexylcarbodiimide
(1.55 g or 1.5 eq.) was added followed by triethylamine (0.7 ml or
1 eq.). The solution was stirred overnight at room temperature, 20
ml of CHCl.sub.3 was added, the solution was washed with 0.1N
NaHCO.sub.3 (3.times.15 ml), and the organic layer was dried and
purified on a silica gel column using step gradient of MeOH in
CH.sub.2Cl.sub.2 to give 137 g (about 64%) of ("28"). Compound 28
(1 g or 233 mmol) was dissolved in 15 ml of 95% EtOH, the solution
cooled to 0.degree. C. and sodium borohydride (176 mg or 4 eq.)
added. The solution was stirred at 0.degree. C. for 1 hour and the
reaction was quenched by addition of 1 ml acetone and neutralized
with 0.1N HCl. The solution was then extracted with 3.times.15 ml
of CHCl.sub.3. Extracts were combined, dried, and purified on a
silica gel column using step gradient of MeOH in CH.sub.2Cl.sub.2
to give 832 mg (about 83%) of compound 29. Compound 29 (1.29 g or 3
mmol) was dissolved in 10 ml of DMF-acetonitrile (1:1).
N,N'-disuccinimidyl carbonate (Fluka Chemical; Ronkonkoma, N.Y.)
(1.15 g or 1.5 eq.) was added which was followed with chloroform.
The solution was washed with 3.times.15 ml of 0.1N NaHCO.sub.3,
solvents were evaporated to dryness and the residue recrystallized
from acetonitrile to give 1.22 g (about 71%) of photocleavable
coumarin NHS ester ("30").
Example 2 Synthesis of Photocleavable Conjugates
PCB-Amino Acids
[0235] PCB-amino acids were prepared by derivatization of the
.alpha.-amino group of the amino acid. Derivatives were prepared
from either PCB chloroformates or their corresponding
N-hydroxysuccinimidyl esters and amino acids in a weakly alkaline
media using a modification of the procedure by Sigler et al.
(Biopolymers 22:2157, 1983; L. Lapatsanis et al., Can. J. Chem.
60:976, 1982; A. Paquet, Can. J. Chem. 60:2711, 1977). This
procedure was also used for the synthesis of
.epsilon.-NH.sub.2-PCB-Lys wherein the .alpha.-amino group of Lys
is protected with Fmoc. PCB-amino acids were also prepared by
carboxyl or hydroxy group derivatization. Briefly, carboxyl
residues of aspartic acid and glutamic acid were esterified using
PCB-OH. .alpha.-amino groups were protected as Fmoc derivatives and
.alpha.-carboxyl groups were protected as t-butyl esters.
Esterfication of the carboxyl side chain was mediated using
dicyclohexylcarbodiimide (DCC) (P. Sieber, Helv. Chim. Acta
60:2711, 1977). Esterification was also carried out by reacting
PCB-chloroformate with the hydroxyl side chains of appropriately
protected threonine or serine residues (M. Bednarek et al., Int. J.
Pept. Prot. Res. 21:196, 1983; H. Kessler et al., Tetrahedron 281,
1983).
[0236] Preparation and photocleavage of PCB-Leucine-Enkephaline:
Leucine-enkephaline (Sigma Chemical; St. Louis, Mo.) (15.5
.mu.mol/ml in 0.1N NaHCO.sub.3, pH 8.0) and PCB-NHS ester (17
.mu.mol/ml in DMF) were mixed and stirred overnight at room
temperature. At this time HPLC analysis showed complete conversion
of Leu-Enk into PCB-Leu-Enk. HPLC analysis was performed on a
Waters System (Waters Chromatography; Marlboro, Mass.) comprising
of U6K injector, 600 Controller, Novapak C.sub.10-Column
(3.9.times.150 mm) and 996 Photodiode Array detector. Tests were
performed using a linear gradient 30-45% of B over 10 minutes
followed by 45% of .beta. isocratic for 10 minutes.
[0237] PCB-Leu Enk (1.93 .mu.mol/ml in phosphate buffer, pH 7.4)
was irradiated with a long-wavelength, UV-lamp (Blak Ray XX-15 UV
lamp; UVP Inc; San Gabriel, Calif.) at a distance of 15 cm
(emission peak 365 nm, lamp intensity=1.1 mW/cm.sup.2 at a distance
of 31 cm). HPLC analysis showed complete photorelease of Leu-Enk
within 5 minutes and confirmed authenticity of the released
material on the basis of retention time and UV spectra.
[0238] PCB-Leu-Enk (10 nmoles) was incubated for 30 minutes in a
suspension of monomeric-avidin agarose beads (15 nmoles). The
suspension was spun-filtered for 3 minutes (16,000.times.). Binding
efficiency was determined at about 94%. Sample was resuspended in
phosphate buffer (2 ml) and irradiated as described. Released
Leu-Enk was assayed using fluorescamine. Emission spectra were
measured on a SLM 48000 fluorimeter using 380 nm excitation
(.lamda.=488 nm). Photorelease of Leu-Enk was quantitated after 5
minutes of illumination.
Example 3 Solid Phase Synthesis of PCB-Polypeptides
[0239] PCB-amino acids were incorporated into polypeptides by
solid-support peptide synthesis. Standard method for employing base
labile fluorenylmethyloxy (Fmoc) group for the protection of
.alpha.-amino function and acid labile t-butyl derivatives for
protection of .alpha.-carboxyl and reactive side chains were used.
Synthesis was carried out on a polyamide-type resin. Amino acids
were activated for coupling as symmetrical anhydrides or
pentafluorophenyl esters (E. Atherton et al., Solid Phase Peptide
Synthesis, IRL Press, Oxford, 1989). The PCB-amino acid for
site-specific incorporation into polypeptide chain was derivatized
with PCB moiety. Side chain PCB-derivatives, like
.epsilon.-amino-Lys, side chain PCB-AA esters of Glu and Asp, and
esters of Ser, Thr and Tyr, were incorporated within the
polypeptide. These PCB-amino acids were stable during solid phase
peptide synthesis, in 20% piperidine/DMF (Fmoc removal) and 1-95%
trifluoroacetic acid (t-Bu, t-Boc removal cleavage of the peptide
from polyamide resin) (E. Atherton et al., Solid Phase Peptide
Synthesis, IRL Press, Oxford, 1989).
Example 4 Detection and Isolation of Nascent Proteins
[0240] Misaminoacylation of tRNA: The general strategy used for
generating misaminoacylated tRNA is shown in FIG. 10 and involves
truncation of tRNA molecules, dinucleotide synthesis,
aminoacylation of the dinucleotide and ligase mediated
coupling.
[0241] Truncated tRNA molecules were generated by periodate
degradation in the presence of lysine and alkaline phosphatase
basically as described by Neu and Heppel (J. Biol. Chem.
239:2927-34, 1964). Briefly, 4 mmoles of uncharged E. coli
tRNA.sup.Lys molecules (Sigma Chemical; St. Louis, Mo.) were
truncated with two successive treatments of 50 mM sodium
metaperiodate and 0.5 M lysine, pH 9.0, at 60.degree. C. for 30
minutes in a total volume of 50 .mu.l. Reaction conditions were
always above 50.degree. C. and utilized a 10-fold excess of
metaperiodate. Excess periodate was destroyed treatment with 5
.mu.l of 1M glycerol. The pH of the solution was adjusted to 85 by
adding 15 .mu.l of Tris-HCl to a final concentration of 0.1 M. The
reaction volume was increased to 150 .mu.l by adding 100 .mu.l of
water. Alkaline phosphatase (15 .mu.l, 30 units) was added and the
reaction mixture incubated again at 60.degree. C. for two hours.
Incubation was followed by ethanol precipitation of total tRNA,
ethanol washing, drying the pellet and dissolving the pellet in 20
.mu.l water. This process was repeated twice to obtain the
truncated tRNA.
[0242] Dinucleotide synthesis was carried out basically as
performed by Hudson (J. Org. Chem. 53:617-24, 1988), and can be
described as a three step process, deoxycytidine protection,
adenosine protection and dinucleotide synthesis.
[0243] Deoxycytidine protection: All reaction were conducted at
room temperature unless otherwise indicated. First, the 5' and 3'
hydroxyl groups of deoxycytidine were protected by reacting with
4.1 equivalents of trimethylsilyl chloride for 2 hours with
constant stirring. Exocyclic amine function was protected by
reacting it with 1.1 equivalents of Fmoc-Cl for 3 hours.
Deprotection of the 5' and 3' hydroxyl was accomplished by the
addition of 0.05 equivalents of KF and incubation for 30 minutes.
The resulting product was produced at an 87% yield. Phosphate
groups were added by incubating this compound with 1 equivalent of
bis-(2-chlorophenyl) phosphorochloridate and incubating the mixture
for 2 hours at 0.degree. C. The yield in this case was 25-30%.
[0244] Adenosine protection: Trimethylsilyl chloride (4.1
equivalents) was added to adenosine residue and incubated for 2
hours, after which, 1.1 equivalents of Fmoc-Cl introduced and
incubation continued for 3 hours. The TMS groups were deprotected
with 0.5 equivalents of fluoride ions as described above. The Fmoc
protected adenosine was obtained in a 56% yield. To further protect
the 2'-hydroxyl compound 22 was reacted with 1.1 equivalents of
tetraisopropyl disiloxyl chloride (TIPDSCl.sub.2) for 3 hours which
produces compound 23 at a 68-70% yield. The compound was converted
to compound 24 by incubation with 20 equivalents of dihydropyran
and 0.33 equivalents of p-toluenesulfonic acid in dioxane for about
4-5 hours. This compound was directly converted without isolation
by the addition of 8 equivalents of tetrabutyl ammonium fluoride in
a mixture of tetrahydro-furan, pyridine and water.
[0245] Dinucleotide synthesis: The protected deoxycytidine,
compound 20 (FIG. 19), and the protected adenosine were coupled by
the addition of 1.1 equivalents of 2-chlorophenyl bis-(1-hydroxy
benzotriazolyl) phosphate in tetrahydrofuran with constant stirring
for 30 minutes. This was followed by the addition of 13 equivalents
of protected adenosine in the presence of N-methylimidazole for 30
minutes. The coupling yield was about 70% and the proton NMR
spectrum of the coupled product is as follows: (.delta. 8.76 m,
2H), (.delta. 8.0 m, 3H), (.delta. 7.8 m, 3H) (.delta. 7.6 m, 4H),
(.delta. 7.5 n, 3H), (.delta. 7.4 m, 18H), (.delta. 7.0 m, 2H),
(.delta. 4.85 m, 14H), (.delta. 4.25 m, 1H); (.delta. 3.6 m, 2H),
(.delta. 3.2 m, 2H) (.delta. 2.9 m, 3H), (.delta. 2.6 m, 1H),
(.delta. 2.0-1.2 m, 7H).
[0246] Aminoacylation of the dinucleotide was accomplished by
linking the N.alpha.-protected N.epsilon.-PCB-lys, to the
dinucleotide with an ester linkage. First, the protected amino acid
was activated with 6 equivalents of benzotriazol-1-yl-oxy
tris-(dimethylamino) phosphonium hexafluoro phosphate and 60
equivalents of 1-hydroxybenzotriazole in tetrahydrofuran. The
mixture was incubated for 20 minutes with continuous stirring. This
was followed with the addition of 1 equivalent of dinucleotide in 3
equivalents N-methylimidazole, and the reaction continued at room
temperature for 2 hours. Deprotection was carried out by the
addition of tetramethyl guanidine and 4-nitrobenzaldoxime, and
continuous stirring for another 3 hours. The reaction was completed
by the addition of acetic acid and incubation, again with
continuous stirring for 30 minutes at 0.degree. C. which produced
the aminoacylated dinucleotide.
[0247] Ligation of the tRNA to the aminoacylated dinucleotide was
performed basically as described by T. G. Heckler et al.
(Tetrahedron 40: 87-94, 1984). Briefly, truncated tRNA molecules
(8.6 O.D..sub.260 units/ml) and aminoacylated dinucleotides (4.6
O.D..sub.260 units/ml), were incubated with 340 units/ml T4 RNA
ligase for 16 hours at 4.degree. C. The reaction buffer included 55
mM Na-Hepes, pH 7.5, 15 mM MgCl.sub.2, 250 .mu.M ATP, 20 .mu.g/ml
BSA and 10% DMSO. After incubation, the reaction mixture was
diluted to a final concentration of 50 mM NaOAc, pH 4.5, containing
10 mM MgCl.sub.2. The resulting mixture was applied to a
DEAE-cellulose column (1 ml), equilibrated with 50 mM NaOAc, pH
4.5, 10 mM MgCa.sub.2, at 4.degree. C. The column was washed with
0.25 mM NaCl to remove RNA ligase and other non-tRNA components.
The tRNA-containing factions were pooled and loaded onto a
BD-cellulose column at 4.degree. C., that had been equilibrated
with 50 mM NaOAc, pH 4.5, 10 mM MgCl.sub.2, and 1.0 M NaCl.
Unreacted tRNA was removed by washes with 10 ml of the same buffer.
Pure misaminoacylated tRNA was obtained by eluting the column with
buffer containing 25% ethanol.
[0248] Preparation of Extract: Wheat Germ Embryo Extract was
Prepared by floatation of wheat germs to enrich for embryos using a
mixture of cyclohexane and carbon tetrachloride (1:6), followed by
drying overnight (about 14 hours). Floated wheat germ embryos (5 g)
were ground in a mortar with 5 grams of powdered glass to obtain a
fine powder. Extraction medium (Buffer I: 10 mM tris-acetate
buffer, pH 7.6, 1 nM magnesium acetate, 90 mM potassium acetate,
and 1 mM DTT) was added to small portions until a smooth paste was
obtained. The homogenate containing disrupted embryos and 25 ml of
extraction medium was centrifuged twice at 23,000.times.g. The
extract was applied to a sephadex G-25 fine column and eluted in
Buffer II (10 mM tris-acetate buffer, pH 7.6, 3 mM magnesium
acetate, 50 mM potassium acetate, and 1 mM DTI). A bright yellow
band migrating in void volume and was collected (S-23) as one ml
fractions which were frozen in liquid nitrogen.
[0249] Preparation of Template: Template DNA was Prepared by
Linearizing plasmid pSP72-bop with EcoRI. Restricted linear
template DNA was purified by phenol extraction and DNA
precipitation. Large scale mRNA synthesis was carried out by in
vitro transcription using the SP6-ribomax system (Promega; Madison,
Wis.). Purified mRNA was denatured at 67.degree. C. for 10 minutes
immediately prior to use.
[0250] Cell-Free Translation Reactions: The incorporation mixture
(100 .mu.l) contained 50 .mu.l of S-23 extract, 5 mM magnesium
acetate, 5 mM tris-acetate, pH 7.6, mM Hepes-KOH buffer, pH 75; 100
mM potassium acetate, 0.5 mM DTT, 0375 mM GTP, 2.5 mM ATP, 10 mM
creatine phosphate, 60 .mu.g/ml creatine kinase, and 100 .mu.g/ml
mRNA containing the genetic sequence which codes for
bacteriorhodopsin. Misaminoacylated PCB-lysine was added at 170
.mu.g/ml and concentrations of magnesium ions and ATP were
optimized. The mixture was incubated at 25.degree. C. for one
hour.
[0251] Isolation of Nascent Proteins Containing PCB-Lysine:
Streptavidin coated magnetic Dynabeads M-280 (Dynal; Oslo, Norway),
having a binding capacity of 101 g of biotinylated protein per mg
of bead. Beads at concentrations of 2 mg/ml, were washed at least 3
times to remove stabilizing BSA The translation mixture containing
PCB-lysine incorporated into nascent protein was mixed with
streptavidin coated beads and incubated at room temperature for 30
minutes. A magnetic field was applied using a magnetic particle
concentrator (MPC) (Dynal; Oslo, Norway) for 0.5-1.0 minute and the
supernatant removed with pipettes. The reaction mixture was washed
3 times and the magnetic beads suspended in 50 .mu.l of water.
[0252] Photolysis was carried out in a quartz cuvette using a
Black-Ray long wave UV lamp, Model B-100 (UV Products, Inc.; San
Gabriel, Calif.). The emission peak intensity was approximately
1100 .mu.W/cm.sup.2 at 365 nm. Magnetic capture was repeated to
remove the beads. Nascent proteins obtained were quantitated and
yields estimated at 70-95%.
Example 5 In Vitro Synthesis of Nascent Proteins using
Photocleavable Conjugates
[0253] Post-Aminoacylation Linkage: A schematic representation of
the steps involved in incorporation of PCB-amino acid for the
detection and/or isolation of targets using post-aminoacylation
linkage is shown in FIG. 10. E. coli tRNA.sup.Lys (Sigma Chem.; St
Louis, Mo.) was aminoacylated with lysine (A. E. Johnson et al.,
Proc. Natl. Acad. Sci. USA 75:3075, 1978). The NHS ester of PCB
(compound 13) dissolved in dimethyl sulphoxide, was added at
0.degree. C. to the solution of Lys-tRNA.sup.Lys and the modified
tRNA purified using benzoylated DEAE-cellulose column (U. C. Kreig
et al., Proc. Natl. Acad Sci. USA 83:8604, 1986). mRNA was
translated in a cell-free, wheat-germ system as described by Sonar
et al. (Biochem. 32:13777, 1993). Nascent proteins containing
PCB-lysine were purified by acetone precipitation to remove
PCB-lysyl tRNA followed by magnetic capture of nascent proteins
containing PCB-lysine using streptavidin coated magnetic beads.
Material obtained after magnetic capture was irradiated for 10
minutes to release nascent protein.
Example 6 Synthesis of Photocleavable Conjugates
PCB Nucleotides
[0254] Synthesis of PCB-dUTP (FIG. 13A): 5-(3-Aminoallyl)-dUTP
ammonium salt ("31") (Sigma Chemical; St. Louis, Mo.) (10 mg or
16.6 .mu.mol) was dissolved in 200 .mu.l of 0.1N NaHCO.sub.3. To
this solution was added a solution of PCB-NHS (compound 13; 12.5 mg
or 1 eq.) in 100 .mu.l of DMF. The reaction mixture was stirred
overnight at room temperature, concentrated, and purified by
reverse-phase semi-preparative HPLC (Novapak C.sub.18 column;
Waters Chromatography; Marlboro, Mass.) using a 10-50% linear
gradient of acetonitrile (B) in 5 mM triethylammonium acetate (A)
over 30 minutes. Fractions containing PCB-dUTP were pooled,
lyophilized, and redissolved in TE buffer (pH 7.4) to a
concentration of 5 nM, and the solution used for enzymatic
incorporation into nucleic acids (yield about 56%). Similar
procedures were used to prepare PCB-UTP, PCB-(d)ATP, and
PCB-(d)CTP, using 5-(3-aminohexyl)-(deoxy)cytidine triphosphate,
respectively.
Example 7 Synthesis of Photocleavable Conjugates
PCB Phosphoramidites
[0255] Synthesis of PCB-phosphoramidite (FIG. 13B):
5-(-5-biotinamidocapro-amidomethyl)-2-nitroacetophenone ("37") (534
mg or 1 mmol) was made anhydrous by coevaporation with pyiridine
(3.times.2 ml) and dissolved in 5 ml of pyridine.
4,4'-dimethoxytrityl chloride (406 mg or 1.2 eq) and
4-dimethylaminopyridine (6 mg or 0.05 eq) were added and the
resulting solution stirred at room temperature for 24 hours. Ten ml
of CHCl.sub.3 and 20 ml of 0.1N aqueous NaHCO.sub.3 were added, the
layers formed separated and the organic layer dried, evaporated to
dryness and purified on a silica gel column using 0-5% step
gradient of MeOH in CHCl.sub.3 to give 576 mg (about 69%) of
compound 38. Intermediate 38 (836 mg or 1 mmol) was dissolved in 8
ml of 95% EtOH. The solution was cooled to 0.degree. C. and
vigorously stirred. To the solution was added NaBH.sub.4 (19 mg or
2 eq.) in portions and the solution stirred for an additional 2
hours at room temperature. The reaction was quenched with 2 ml of
acetone, 10 ml of CHCl.sub.3 and 10 ml of 0.1N aqueous NaHCO.sub.3
were added, the layers were separated, the organic layer was dried
and evaporated to dryness to give 704 mg (about 84%) of compound 39
which was used without additional purification. Compound 39 (838 mg
or 1 mmol) was dissolved in a mixture of CHCl.sub.3 (5 ml) and
diisophrophylethylamine (0.68 ml or 4 eq.). To this solution was
added 2-cyanoethyl-N-,N-diisophropylchlora-phosphoramidite (225
.mu.l or 1 eq.) and the solution was stirred at room temperature
for 1 hour. Ethyl acetate (5 ml) was added and the solution was
washed with an NaCl solution (3.times.1 ml) and H.sub.2O (2 ml).
Dried solvents were removed in vacuo and purified on a silica gel
column using step gradient of triethylamine in CH.sub.2Cl.sub.2
with a yield of 789 mg (about 74%).
Example 8 Chemical Synthesis of Oligonucleotides using
PCB-Phosphoramidites
[0256] Automated synthesis and purification of oligonucleotides
using 5'PCB-phosphoramidite: A 0.1M solution of
5'-PCB-phosphoramidite in anhydrous acetonitrile was prepared. The
bottle with the solution was placed in the additional
phosphoramidite port of the Applied Biosystem 392 DNA/RNA
synthesizer. An oligodeoxynucleotide sequence was programmed and
synthesized using 40 nmol CE column and standard synthesis
protocol. The only modification necessary was extended
detritylation (180 s) necessary for removal of trityl group from
N.sup.1 position of biotin. After synthesis,
5'-PCB-oligodeoxynucleotide was cleaved from solid support and
deprotected by treatment with concentrated ammonia for 16 hours at
50.degree. C. The crude oligonucleotide was freeze-dried and
dissolved in 1 ml of phosphate buffer, pH 7.4. To this solution was
added a suspension of monomeric avidin agarose beads (Sigma
Chemical; St. Louis, Mo.) (40 nmoles), and the mixture was
incubated at room temperature for 1 hour. The suspension was
filtered and washed with 3.times.1 ml phosphate buffer, resuspended
in 3 ml of phosphate buffer and illuminated as described with
gentle stirring for 10 minutes. The mixture was filtered, filtrate
freeze-dried and redissolved (yield=3.8 OD.sub.260).
Example 9 Enzymatic Synthesis of DNA and RNA using
PCB-Nucleotides
[0257] Several enzymatic and chemical methods are available for
biotinylation of nucleic acid probes. Enzymatic methods for
incorporation of PCB-nucleotides into DNA include nick translation
and replacement synthesis using T4 DNA polymerase. Terminal
labeling of DNA can also be performed using terminal
deoxynucleotidyl transferase. For PCB-labeling of RNA several RNA
polymerase enzymes can be used. Nick translation was performed in
the presence of PCB-dCTP based on the methods developed for
biotinylation (P. R. Langer et al., Proc. Natl. Acad. Sci. USA
78:6633, 1981). Enzymatic tailing was used for double- and
single-stranded DNA molecules. PCB nucleotides were added onto the
3'-end of the DNA. Biotinylated probes with internal biotin
moieties form less stable hybrids than probes with external biotins
and that the biotinylated probes synthesized in this manner have
greater sensitivity than probes that are singly biotinylated at
5'-end (E. P. Diamandis et al., Clin. Chem. 37:625, 1991).
[0258] Preparation of PCB-Labeled RNA: PCB-Labeling of RNA was
Achieved in a standard phage T7 RNA polymerase transcription system
using the PCB-UTP. To prepare single-stranded, biotinylated RNA as
a probe, the appropriate DNA sequence was cloned into an
appropriate vector which contains the 17 promoter upstream from the
polylinker region. After linearization of the DNA clone downstream
from the cloned insert, the RNA transcript of defined length was
produced by the T7 RNA polymerase using ATP, CTP, GTP and PCB-UTP
as substrates.
Example 10 Isolation of Hematopoietic Cells for Autologous Bone
Marrow Transplantation
[0259] Bone marrow is collected from the posterior iliac crest of
normal healthy and leukemic patients into heparin. Low-density
mononuclear cells are separated by sedimentation on Ficoll-Hypaque
(Sigma Chemical; St Louis, Mo.). CD34.sup.+ cells are isolated
using PCB-labeled anti-CD34 monoclonal antibodies (My10).
Mononuclear marrow cells are placed at concentrations of
10.sup.6/ml in Iscove's Modified Dulbeco's Medium (IMDM; Irvine
Scientific; Santa Anna, Calif.) with 20% FCS. Cells are cultured
overnight under tissue culture conditions to remove adherent cells.
Nonadherent cells are collected, washed twice in cold phosphate
buffered saline (PBS), and diluted in PBS to 10.sup.7/ml.
PCB-labeled anti-CDM34 antibodies are added to the cell suspension
at 5 .mu./ml and incubated at 4.degree. C. for one hour with gentle
intermittent mixing. After incubation, cells are washed twice in
5%-FCS/PBS and resuspended in the same volume. Streptavidin coated
magnetic beads (Dynabeads; Oslo, Norway) are added to the
suspension which is incubated at 4.degree. C. for one hour with
mixing. Beads and their associated cells are subjected to a magnet
and separated from the suspension and placed in 5%-FCS/PBS.
Photocleavage is carried out by irradiating the beads for 4 minutes
with a long-wavelength, UV-lamp (Black Ray XX-15 UV lamp; UVP Inc;
San Gabriel, Calif.) at a distance of 15 cm (emission peak 365 nm,
lamp intensity=1.1 mW/cm.sup.2 at a distance of 31 cm). Released
beads are isolated by magnetic capture. The cell suspension is
assayed for CD34.sup.+ cells by staining with FITC-conjugated My10
antibody followed with FACS analysis and determined to be greater
than 95% CD34 cells.
Example 11 Determination of the In Vivo Half-life of a
Pharmaceutical Composition
[0260] Cell-free translation reactions are performed by mixing 10
.mu.l of PCB-coumarin amino acid-tRNA.sup.leu, prepared by chemical
misaminoacylation as described above and suspended in TE at 1.7
mg/ml), 50 .mu.l of S-23 extract, 10 .mu.l water and 10 .mu.l of a
solution of 50 mM magnesium acetate, 50 mM Tris-acetate, pH 7.6,
200 mM Hepes-KOH buffer, pH 7.5; 1 M potassium acetate, 5 mM DTT,
3.75 mM GTP, 25 mM ATP, 100 mM creatine phosphate and 600 .mu.g/ml
creatine kinase. This mixture is kept on ice until the addition of
20 .mu.l of 500 .mu.g/ml human IL-2 mRNA (containing 26 leucine
codons) transcribed and isolated from recombinant IL-2 cDNA. The
mixture is incubated at 25.degree. C. for one hour and placed on
ice. One hundred .mu.l of streptavidin coated magnetic Dynabeads (2
mg/ml) are added to the mixture which is placed at room temperature
for 30 minutes. After incubation, the mixture is centrifuged for 5
minutes in a microfuge at 3,000.times.g or, a magnetic field is
applied to the solution using a MPC. Supernatant is removed and the
procedure repeated three times with TE. The final washed pellet is
resuspended in 50 .mu.l of 50 mM Tris-HCl pH 7.5 and transferred to
a quartz cuvette. UV light from a Black-Ray long wave UV lamp is
applied to the suspension for approximately one second. A magnetic
field is applied to the solution with a MPC for 1.0 minute and the
supernatant removed with a pipette. The supernatant is sterile
filtered and mixed with equal volumes of sterile buffer containing
50% glycerol 1.8% NaCl and 25 mM sodium bicarbonate. Protein
concentration is determined by measuring the O.D..sub.260.
[0261] 0.25 ml of the resulting composition is injected iv. into
the tail vein of 2 Balb/c mice at concentrations of 1 mg/ml. Two
control mice are also injected with a comparable volume of buffer.
At various time points (0, 5 minutes, 15 minutes, 30 minutes, 60
minutes, 2 hours and 6 hours), 100 .mu.l serum samples are obtained
from foot pads and added to 400 .mu.l of 0.9% saline. Serum sample
are added to a solution of dynabeads (2 mg/ml) coated with
anti-coumarin antibody and incubated at room temperature for 30
minutes. A magnetic field is applied to the solution with a MPC for
1 minute and the supernatant removed with a pipette. Fluorescence
at 470 nm is measured and the samples treated with monoclonal
antibody specific for rat IL-2 protein. IL-2 protein content is
quantitated for each sample and equated with the amount of
fluorescence detected. From the results obtained, in vivo IL-2
half-life is accurately determined.
Example 12 Polymerase Chain Reactions with PCB
[0262] The steps involved in the PCR amplification DNA sequences
using PCB are shown in FIG. 12. The experimental method described
below is based on a combination of protocols described (Y. Lo et
al., Nucl. Acids Res. 16:719, 1988; R. K. Saiki et al., Sci
239:487, 1988). PCB-dCTP synthesized was added (50 .mu.M) to the
mixture of dATP, dGTP, dTTP (all 200 .mu.M) and dCTP (150 .mu.M).
The reaction mixture consisted of target DNA source (total genomic
DNA isolated), flanking primers and the thermostable polymerase
(Taq polymerase). The reaction mixture was subjected to 25-30
cycles of amplification. Samples were heated from 70-95.degree. C.
for a 1 minute period to denature DNA and cooled to 40.degree. C.
for 2 minutes to anneal the primers. Samples were again heated to
70.degree. C. for 1 minute to activate the polymerase and incubated
at this temperature for 0.5 minutes to extend the annealed primers.
After the last cycle, samples were incubated for an additional 5 to
10 minutes at 37.degree. C. to ensure that the final extension was
complete. Magnetic capture of the nucleic acids was performed using
streptavidin coated magnetic beads. The captured material was
washed with appropriate buffers and resuspended at the desired
concentration. Samples were illuminated for 10 minutes to release
the PCR product in an unmodified form.
[0263] Nucleic acids in either immobilized form or in solution form
were detected or separated (purified) using PCB-labeled nucleic
acid probes. Hybridization was carried out to obtain DNA:DNA,
DNA:RNA and RNA:RNA hybrids. These experiments involve first the
hybridization with PCB-labeled probes followed by capturing the
hybrids using streptavidin coated immobilized supports. These
hybrids were washed free of initial undesired components and were
released from the immobilized support using irradiation (G.
Gebeyehu et al., Nucl. Acids Res. 15:4513, 1987; T. Ito et al.,
Nucl. Acids Res. 20:3524, 1992). Hybridization of ssDNA molecules
with PCB-probe involved incubation of these components in a
hybridization buffer at 42.degree. C. for 30 minutes. Hybridization
conditions were optimized for each probe and experimental system.
PCB-labeled probes had lower melting temperatures than radiolabeled
probes and require slightly modified hybridization conditions.
These hybrids were selectively removed from the reaction components
using immobilized streptavidin (Dynabeads M280 streptavidin).
Photochemical release of complexes resulted in the isolation of
pure hybrid.
Example 13 Synthesis of PCB
Liposomes
[0264] Incorporation of PCB-Lipids into liposomes: PCB-lipids were
mixed with conventional lipids in chloroform:methanol at a ratio of
2:1. The lipid mixture was evaporated to dryness under nitrogen and
the dried lipids suspended in DMSO as solvent to a final
concentration of 1 mg/ml. Liposomes were sonication under nitrogen
in an ice-cold chamber for 10 minutes. The resulting suspension was
centrifuged for 20 minutes at 10,000 rpm and the supernatant
containing PCB-liposomes was ready for use. Satisfactory results
were obtained with as little as 5% (mol equivalent) PCB-lipids. The
structural chemical formulas for PCB-phosphatidylathanolamine and
PCB-phosphatdylserine are shown in FIG. 16.
Example 14 PCB for In Situ Hybridization
[0265] The general methodology for in situ hybridization reactions
can be divided into sample preparation, selection of indicator
molecule and probe, hybridization, washing, and autoradiography and
detection.
[0266] Sample preparation: Frozen tissue sections of 5 to 6 .mu.m
are mounted on gelatin coated microscope slides and air dried for
30 min prior to fixation. This is followed by fixation of DNA or
RNA using either glutaraldehyde or paraformaldehyde. During these
fixing steps optional denaturing steps (e.g. 100.degree. C. for 5
minutes) followed by quick immersion in ice cold buffer (necessary
if dsDNA or dsRNA is the target of the reaction) can be introduced.
In case of cells and cell-cultures, these cells (1.times.10.sup.6
cells) are deposited on gelatin coated slides by cytocentrifugation
or smearing. The cells are air dried and fixed for DNA or RNA.
[0267] Selection of indicator molecule and probe: Although isotopic
detection offers several advantages over the use of non-isotopic
methods, the latter can be used effectively. Labeled probes can be
generated by a variety of techniques ranging from synthetic
oligonucleotides to excised plasmid inserts.
[0268] Hybridization: ISH follows the same general principles as a
solution and filter hybridization. Standard reaction temperatures
are approximately T.sub.m-25.degree. C. (T.sub.m is the temperature
at which 50% of hybrids dissociate). The reaction temperature is
reduced to a level compatible with the preservation of histological
detail by the addition of 50% formamide to the hybridization
mixture. Thus, for typical DNA-DNA hybridization reactions, the
temperature is 37.degree. C., for RNA-DNA 44.degree. C., and for
RNA-RNA 50.degree.. The surface of the microscope slide supporting
the sample is gently blown by a stream of air, and the final
hybridization mix is pipetted over the surface. The film is then
incubated flat in a bath of paraffin oil for the required time and
temperature.
[0269] Washing: The paraffin oil is drained and excess of oil is
removed by washing twice with chloroform, and the slides are air
dried. A high stringency wash is given to reduce background.
[0270] Autoradiography and detection: Detection is carried out
either using X-ray film or emulsion coated cover-slips in cases of
radioactive isotopically labeled probes, and other methods in the
case of enzymatic detection methods.
Example 15 Isolation of Different Populations Cells with Agents
which Photocleaved at Distinct Wavelengths
[0271] Two distinct conjugates are created, each with a different
antigen-specific antibody coupled to a different bioreactive agent.
Conjugate A comprises compound 30 (FIG. 21), a PCB bioreactive
agent, coupled to an antibody specific for the cell surface marker
CD34 (a stem cell marker), and will photocleave with radiation at
300 nm. Conjugate B comprises compound 25 (FIG. 19), a PCB
bioreactive agent, coupled to an antibody specific for the cell
surface marker CD3 (a T cell marker), and will photocleave with
radiation at 400 nm.
[0272] Conjugates A and B are incubated, in duplicate, with samples
of peripheral blood obtained from healthy human volunteers.
Incubations are performed at room temperature (22.degree. C.) with
gentle rocking to provide maximal antibody-antigen contact. After a
30 minute incubation, cells are placed in 100 mm tissue culture
dishes coated with streptavidin and incubated for an additional 30
minutes. Upon streptavidin-biotin binding, plates are gently washed
in PBS to remove any cells which do not adhere.
[0273] After washing, one set of plates is treated with
electromagnetic radiation at 300 nm and the released cells
collected. This set is then treated with electromagnetic radiation
at 400 nm and the cells released at this frequency collected. A
second set of plates is treated with radiation at 400 nm, released
cells are collected, the plates are again treated at 300 nm and the
released cells again collected. By determining the number of cells
collected after each treatment and from each set of plates, the
number of cells in a sample of peripheral blood which carry the
cell surface marker for CD34, CD3, and both CD34 and CD3 is
determined.
[0274] Other embodiments and uses of the invention will be apparent
to those skilled in the art from consideration of the specification
and practice of the invention disclosed herein. It is intended that
the specification and examples be considered exemplary only, with
the true scope and spirit of the invention being indicated by the
following claims.
Sequence CWU 1
1
1111DNAArtificial SequenceSynthetic 1aaaaaagggg g 11
* * * * *